Chemoenzymatic methods for the synthesis of statins and statin intermediates

ABSTRACT

The invention provides aldolases, nucleic acids encoding them and methods for making and using them, including chemoenzymatic processes for making β,δ-dihydroxyheptanoic acid side chains and compositions comprising these side chains, e.g., [R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoic acid (atorvastatin, LIPITOR™), rosuvastatin (CRESTOR™), fluvastatin (LESCOL™), related compounds and their intermediates.

RELATED APPLICATIONS

This application is divisional of U.S. patent application Ser. No.10/472,157, filed Aug. 19, 2003 (Intl.), now pending, which is anational phase application claiming benefit of priority under 35 U.S.C.§371 to Patent Convention Treaty (PCT) International Application SerialNo: PCT/US03/27334, filed Aug. 19, 2003, which claims benefit ofpriority to U.S. Provisional Patent Application Ser. No. 60/412,625,filed Sep. 20, 2002, and U.S. Ser. No. 60/469,374, filed May 9, 2003.The aforementioned applications are explicitly incorporated herein byreference in their entirety and for all purposes.

SEQUENCE LISTING

This application is being filed electronically via the USPTO EFS-WEBserver, as authorized and set forth in MPEP §1730 II.B.2(a)(A), and thiselectronic filing includes an electronically submitted sequence (SEQ ID)listing. The entire content of the sequence listing is hereinincorporated by reference for all purposes. The sequence listing isidentified on the electronically filed .txt file as follows:

Date of File Name Creation Size (bytes) 564462008810SEQLIST.txt02/08/2008 44,304 bytes

TECHNICAL FIELD

This invention relates to the field of synthetic organic and medicinalchemistry, and pharmaceuticals. In particular, the invention providesnovel aldolases, nucleic acids encoding them and methods for making andusing them, including chemoenzymatic processes for makingβ,δ-dihydroxyheptanoic acid side chains and compositions comprisingthese side chains, e.g., (R)-ethyl-4-cyano-3-hydroxybutyrate(atorvastatin, LIPITOR™), rosuvastatin (CRESTOR™), fluvastatin(LESCOL™), related compounds, e.g., statins, and their intermediates.

BACKGROUND

The importance of chiral drugs in the pharmaceutical market increaseswith each year. Single stereoisomers on the market have proven to besafer, exhibit fewer side effects, and are more potent than what achiraldrugs have been previously able to afford. The fact that pharmaceuticalcompanies can now consider the practicality of marketing chiral drugs ispartially due to the ability of synthetic chemists to be able to obtainhigh enantiomeric excess in asymmetric bond construction.

[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, LIPITOR™), whose structure is set forth in FIG. 5,belongs to a class of drugs called statins. Statins reduce the level oftotal cholesterol and LDL by inhibiting HMG-CoA reductase, an enzymethat catalyzes the conversion of HMG-CoA to mevalonate. Atorvastatin isthe most potent of the statins. Atorvastatin contains a chiralβ,δ-dihydroxyheptanoic acid side chain that requires a significanteffort to produce on a large scale. Fluvastatin (LESCOL™) is watersoluble and acts through the inhibition of3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase.

The aldol addition reaction, or aldol condensation, is a fundamentalorganic chemistry method for the formation and dissociation ofcarbon-carbon bonds. The aldol condensation can create two contiguousstereogenic centers and, consequently, four stereoisomers. Some controlover the stereoselectivity can be obtained by the use of preformedenolates with metals. However, these reagents are stoichiometric andrequire extensive protecting group chemistry. See, for example, C. H.Heathcock, Aldrichim. Acta (1990): vol. 23, p 99; C. H. Heathcock,Science (1981): vol. 214, p 395; D. A. Evans, Science (1988): vol. 240,p 420; S. Masamune, et al., Angew. Chem. Int. Ed. Engl. (1985): vol. 24,p 1; D. A. Evans, et al., Top. Stereochem. (1982): vol. 13, p 1; C. H.Heathcocket et al., in Comprehensive Organic Synthesis, B. M. Trost, Ed.(Pergamon, Oxford, 1991), vol. 2, pp. 133-319 (1991); and I. Paterson,Pure & Appl. Chem. (1992): vol. 64, 1821.

Enantioselectivity can be obtained by using either chiral enolderivatives, chiral aldehydes or ketones, or both. However, recentstudies of catalytic antibodies opened ways to obtain enantiomericallypure aldols via resolution. Thus, for some reactions, the problem ofcomplex intermediates may be solved by using relatively reactivecompounds rather than the more usual inert antigens to immunize animalsor select antibodies from libraries such that the process of antibodyinduction involves an actual chemical reaction in the binding site. See,for example, C. F. Barbas III, et al., Proc. Natl. Acad. Sci. USA(1991): vol. 88, p 7978 (1991); K. D. Janda et al., Proc. Natl. Acad.Sci. USA (1994): vol. 191, p 2532. This same reaction then becomes partof the catalytic mechanism when the antibody interacts with a substratethat shares chemical reactivity with the antigen used to induce it.

The mechanisms of aldol condensation by aldolases have been wellcharacterized. C. Y. Lai, et al., Science (1974): vol. 183, p 1204; andA. J. Morris e al., Biochemistry (1994) vol. 33, p 12291. The enzyme2-deoxyribose-5-phosphate aldolase (DERA) in vivo catalyzes reversiblealdol reaction of acetaldehyde and D-glyceraldehyde 3-phosphate to formD-2-deoxyribose-5-phosphate, the sugar moiety of DNA. Consequently thistype I aldolase is widespread in nature. It is the only aldolase thataccepts two aldehydes as substrates. Recent studies show that, incertain DERA-catalyzed reactions, product of the first aldolcondensation can become an acceptor substrate for a second aldolcondensation catalyzed by DERA or another aldolase. Thus, DERA and otheraldolases can be used in combination for sequential aldol reactionsleading to products with multiple chiral centers, starting from simple,non-chiral substrates. Gijsen, H., Wong, C.-H., JACS, vol. 117,7585-7591. This enzyme can provide a route to a wide range ofpotentially biologically active compounds, e.g., the synthesis ofdeoxysugars such as deoxyriboses, 2-deoxyfucose analogs, and13C-substituted D-2-deoxyribose-5-phosphate. See, for example, U.S. Pat.No. 5,795,749. It also affords a route to a variety of chiral aldehydesas illustrated in FIG. 6.

SUMMARY

The invention provides chemoenzymatic processes for makingβ,δ-dihydroxyheptanoic acid side chains and compositions comprisingthese side chains, e.g., statins. The invention provides methods for theenantioselective assembling of chiral β,δ-dihydroxyheptanoic acid sidechains, including compositions comprising β,δ-dihydroxyheptanoic acidside chain cores, e.g., statins, such as[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, LIPITOR™), rosuvastatin (CRESTOR™), fluvastatin(LESCOL™), related compounds and their intermediates. In one aspect, themethods provide an enantioselective synthesis of both stereogeniccenters of atorvastatin and/or rosuvastatin, and β,δ-dihydroxyheptanoicacid side chain-containing intermediates, in a single transformationfrom low-cost starting materials.

The invention provides methods for preparation of a compound having aformula as set forth as intermediate II in FIG. 7, comprising thefollowing steps: (a) providing an aldol donor substrate; (b) providingan aldol acceptor substrate; (c) providing an aldolase; (d) admixing thealdol donor substrate of step (a), the aldol acceptor substrate of step(b), and the aldolase of step (c) under conditions wherein the aldolasecan catalyze the aldol condensation reaction between the substrates ofsteps (a) and (b) thereby producing a compound comprising a structure asset forth as intermediate II in FIG. 7. In one aspect, the aldolacceptor substrate comprises an aldehyde. In one aspect, the aldehydealdol acceptor substrate comprises a structure as set forth as aldehydeIII in FIG. 7. In one aspect, R in the aldehyde III of FIG. 7 isselected from the group consisting of a hydrogen group, an alkyl group,a C1-C4 alkoxy group, a halogen, a cyan group and an azido group. In oneaspect, R in the aldehyde III of FIG. 7 is chlorine and aldehyde III ischloroacetaldehyde.

In one aspect the method further comprises converting the intermediateII in FIG. 7 to a compound comprising a β,δ-dihydroxyheptanoic acid sidechain. In one aspect, the compound comprising a β,δ-dihydroxyheptanoicacid side chain comprises a structure as set forth in formula I of FIG.7. In one aspect, the aldolase is a 2-deoxyribose-5-phosphate aldolase(DERA), e.g., a recombinant 2-deoxyribose-5-phosphate aldolase (DERA).In one aspect, the aldolase comprises a polypeptide as set forth in SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, or SEQ ID NO:30, or an enzymatically activefragment thereof. In one aspect, the aldolase comprises a polypeptide ofthe invention, or, a polypeptide encoded by a nucleic acid of theinvention.

In one aspect, the aldol donor substrate comprises an acetaldehyde. Inone aspect, the aldol donor substrate comprises an acetaldehyde and thealdol acceptor substrate comprises an aldehyde. In one aspect, theacetaldehyde is present in stoichiometric excess over the aldehyde. Inone aspect, the reaction of step (d) is carried out in the absence oflight. In one aspect, the reaction of step (d) is carried out at atemperature comprising a range from about 5° C. to about 45° C. and a pHvalue of about 6.5 to 8.5.

In one aspect, the method further comprises converting the intermediateII in FIG. 7 to a lactone compound. In one aspect, the lactone is achloro-lactone, e.g., a 6-chloro-2,4,6-trideoxyerythro-hexonolactone. Inone aspect, the lactone is crystalline. In one aspect, the crystallinelactone is purified by recrystallization.

In one aspect, the formation of6-chloro-2,4,6-trideoxyerythro-hexonolactone (chloro-lactone VI in FIG.9) is carried out under oxidation conditions, e.g., comprising bromine(Br₂), BrCO₃ and water, a bromine/barium carbonate oxidation, asillustrated in FIG. 9. In one aspect, the method comprises abromine/barium carbonate oxidation with sodium hypochlorite (NaOCl) inacetic acid (HOAc) and water.

In one aspect, the method further comprises converting the lactonecompound to a compound as set forth as intermediate VIII in FIG. 10. Inone aspect, the method further comprises converting the chloro-lactoneto a compound set forth as lactone IX of FIG. 10. In one aspect, thechloro-lactone is converted to a compound set forth as lactone IX ofFIG. 10 by subjecting the chloro-lactone to a cyanide displacement underconditions wherein the chloro group of the lactone is replaced by a cyangroup CN.

In one aspect, the method further comprises converting the lactone IX toan intermediate VII of FIG. 10. In one aspect, the lactone IX isconverted to an intermediate VII of FIG. 10 under conditions comprisingtreatment with MeOH and Dowex or MeOH and K₂CO₃, wherein the lactonering opens and the intermediate VII is formed. In one aspect, the methodfurther comprises further comprising converting the intermediate VII toan intermediate VIII of FIG. 10. In one aspect, the method furthercomprises processing the lactone to a compound comprising formula I ofFIG. 7.

In one aspect, all reactions occur in a single reaction vessel. In oneaspect, the intermediate II in FIG. 7 is a chloro-substitutedintermediate having a structure as set forth as intermediate II in RouteI, FIG. 8. In one aspect, the intermediate II in Route I, FIG. 8 isconverted to a lactone by a process comprising CN-displacement, lactaloxidation and nitrile reduction.

In one aspect, the intermediate II in Route I, FIG. 8 is converted to alactone by a process comprising bromine/barium carbonate oxidation to achlorolactone. The method using bromine/barium carbonate oxidation cancomprise oxidation with sodium hypochlorite (NaOCl) in acetic acid(HOAc) and water, as illustrated in FIG. 9.

In one aspect, the intermediate II in FIG. 7 is a cyan-substitutedintermediate having a structure as set forth as intermediate II in RouteII, FIG. 8. In one aspect, the intermediate II in Route II, FIG. 8 isconverted to a lactone by a process comprising lactal oxidation andnitrile reduction. In one aspect, the intermediate II is anN₃-substituted intermediate having a structure as set forth asintermediate II in Route III, FIG. 8. In one aspect, the intermediate IIin Route III, FIG. 8 is converted to a lactone by a process comprisinglactal oxidation and azide reduction.

In one aspect, the method further comprises oxidation of the compoundcomprising intermediate II in FIG. 7, wherein R is a halogen, to make acompound comprising 3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone(formula I in FIG. 14). In one aspect, the oxidation conditions compriseCN— displacement, lactal oxidation and nitrile oxidation. In one aspect,R is a chlorine.

In one aspect, the method further comprises processing the3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone to make(3R,5R)-6-cyano-3,5,-dihydroxyhexanoic acid (compound I of FIG. 14). Inone aspect, the process comprises ring-opening. In one aspect, theprocess comprises ring-opening with cyanide. In one aspect, the methodfurther comprises processing (3R,5R)-6-cyano-3,5,-dihydroxyhexanoic acid(compound I of FIG. 14) to make[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, LIPITOR™). In one aspect, the method furthercomprises processing the3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone to make(3R,5S)-3,5,6-trihydroxyhexanoic acid (compound II of FIG. 14). In oneaspect, the process comprises nucleophilic displacement. In one aspect,the nucleophilic displacement process comprises use of a hydroxide,e.g., sodium hydroxide. In one aspect, the method further comprisesprocessing (3R,5S)-3,5,6-trihydroxyhexanoic acid (compound II of FIG.14) to make a rosuvastatin (CRESTOR™). In one aspect, the method furthercomprises processing (3R,5S)-3,5,6-trihydroxyhexanoic acid (compound IIof FIG. 14) to make fluvastatin (LESCOL™).

The invention provides processes for making[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, LIPITOR™) comprising a process as set forth in FIG.14, FIG. 17 and FIG. 18. FIG. 18 illustrates a process of the inventioncomprising a chemoenzymatic route to make an atorvastatin (LIPITOR™)intermediate. The invention provides a process for making compound I ofFIG. 18 using a DERA, e.g., using a DERA of the invention, using aprocess as set forth in FIG. 18. The invention provides a process formaking compound II of FIG. 18 using a DERA, e.g., using a DERA of theinvention, using a process as set forth in FIG. 18. The inventionprovides a process for making compound III of FIG. 18 using a DERA,e.g., using a DERA of the invention, using a process as set forth inFIG. 18. The invention provides a process for making compound II of FIG.18 from compound I of FIG. 18 using dimethyloxypropane, MeOH and H₂SO₄,as set forth in FIG. 18. The invention provides a process for makingcompound III of FIG. 18 from compound II of FIG. 18 using H₂, RaneyNickel, 7N NH₃ at 46° C., as set forth in FIG. 18. These are a conciseand simple syntheses from inexpensive materials.

The invention provides processes for making rosuvastatin (CRESTOR™)comprising a process as set forth in FIG. 14 and FIG. 17. The inventionprovides processes for making rosuvastatin (CRESTOR™) and fluvastatin(LESCOL™) comprising a process as set forth in FIG. 17.

The invention provides methods for preparation of a compound having aformula as set forth as intermediate II in FIG. 7, using a fed-batchprocess, comprising the following steps: (a) providing an aldol donorsubstrate; (b) providing an aldol acceptor substrate; (c) providing analdolase; (d) admixing the aldol donor substrate of step (a), the aldolacceptor substrate of step (b), and the aldolase of step (c) underconditions wherein the aldolase can catalyze the aldol condensationreaction between the substrates of steps (a) and (b), wherein thesubstrates are fed into the reaction over about at least about 30minutes to about 12, 15, 18, 21, 24 or more hours at a rate such thatthey are consumed as fast as they are added. In one aspect, one of thesubstrates is chloroacetaldehyde, and the substrates are fed into thereaction at a rate such that they are consumed as fast as they are addedand the chloroacetaldehyde does not reach inhibitory concentration. Inone aspect, the substrates are fed into the reaction over a time rangeof about 1 to 10 hours, or, about 2 to 8 hours, or, about 2 to 4 hours,or, about 2 to 3 hours. In one aspect, the method further comprisesprocessing intermediate II as in FIG. 7 to make an atorvastatin(LIPITOR™). In one aspect, the method further comprises processingintermediate II as in FIG. 7 to make a rosuvastatin (CRESTOR™) and/orfluvastatin (LESCOL™). In one aspect, the aldolase is a2-deoxyribose-5-phosphate aldolase (DERA), e.g., a recombinant2-deoxyribose-5-phosphate aldolase (DERA). In one aspect, the aldolasecomprises a polypeptide as set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30, or an enzymatically active fragment thereof. In one aspect, thealdolase comprises a polypeptide of the invention, or, a polypeptideencoded by a nucleic acid of the invention.

The invention provides methods for making3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone (compound 1 of FIG.14) comprising oxidation of a chlorolactol to a chlorolactone withsodium hypochlorite. In one aspect, the chlorolactone comprises acrystalline chlorolactone. In one aspect, the chlorolactol comprises acrude chlorolactol. In one aspect, the chlorolactol is dissolved inglacial acetic acid, and about 1 equivalent of aqueous sodiumhypochlorite is fed into the solution. In one aspect, about 1 equivalentof aqueous sodium hypochlorite is fed into the solution over about 3hours.

The invention provides methods for making3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone (compound 1 of FIG.14) comprising a process as set forth in FIG. 14 and/or FIG. 15.

The invention provides methods for making an epoxide(-(3R,5S-3-hydroxy-4-oxiranylbutyric acid) (structure 2 in FIG. 16)using a process as set forth in FIG. 16. In one aspect, the methodcomprises use of NaCN (e.g., 3 equivalents of NaCN), dimethylformamide(DMF) and water (e.g., 5% H₂O, DMF with 5% water by volume). In anotheraspect, the method comprises use of 2.2 equivalents of NaCN, water(e.g., 5% H₂O) at about 40° C., for about 20 hours. These processes cangenerate the intermediate (3R,5R)-6-cyano-3,5,-dihydroexyhexanoic acid(a protected side chain intermediate). In one aspect, this is a one-potprocess. In one aspect of the reaction in FIG. 16 and FIG. 18, thelactone ring is opened and chloride is displaced by hydroxide, againthrough the epoxide intermediate, to access the trihydroxy acid. Thereaction conditions can comprise 2 equivalents of sodium hydroxide inwater.

The invention provides methods for making(3R,5S)-3,5,6-trihydroxyhexanoic acid comprising a process as set forthin FIG. 16 and FIG. 18, e.g., through the epoxide intermediate-(3R,5S-3-hydroxy-4-oxiranylbutyric acid. In one aspect, the processcomprises use of water and NaOH. In one aspect, this is a one-potprocess.

In one aspect, the invention provides a one pot process to make statinintermediates comprising a lactone opening and a cyanide displacementthrough epoxide intermediates (e.g., -(3R,5S-3-hydroxy-4-oxiranylbutyricacid, structure 2 in FIG. 16), as set forth in FIG. 16 and FIG. 18. Inone aspect, the invention provides a one pot process for making(3R,5S)-3,5,6-trihydroxyhexanoic acid comprising a process as set forthin FIG. 16 and FIG. 18. The methods can further comprise synthesis ofatorvastatin (LIPITOR™), rosuvastatin (CRESTOR™), fluvastatin (LESCOL™)and related compounds. A complete exemplary process for the synthesis ofstatin intermediates (for, e.g., synthesis of atorvastatin (LIPITOR™),rosuvastatin (CRESTOR™), fluvastatin (LESCOL™) and related compounds) isillustrated in FIG. 21. In alternative aspects various steps of theprocess, or the entire process, is a one-pot process.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ IDNO:5 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, ormore residues, encodes at least one polypeptide having aldolaseactivity, and the sequence identities are determined by analysis with asequence comparison algorithm or by a visual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ IDNO:7 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, ormore residues, encodes at least one polypeptide having aldolaseactivity, and the sequence identities are determined by analysis with asequence comparison algorithm or by a visual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQID NO:9 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,or more residues, encodes at least one polypeptide having aldolaseactivity, and the sequence identities are determined by analysis with asequence comparison algorithm or by a visual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQID NO:11 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,or more residues, encodes at least one polypeptide having aldolaseactivity, and the sequence identities are determined by analysis with asequence comparison algorithm or by a visual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity to SEQ ID NO:13 over a region of at least about 10, 15, 20, 25,30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, or more residues, encodes at least one polypeptidehaving aldolase activity, and the sequence identities are determined byanalysis with a sequence comparison algorithm or by a visual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to SEQ ID NO:15 over a region of atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, encodesat least one polypeptide having aldolase activity, and the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by a visual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to SEQ ID NO:17 over a region of at least about 10,15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, or more residues, encodes at least onepolypeptide having aldolase activity, and the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to SEQ ID NO:19 over a regionof at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues,encodes at least one polypeptide having aldolase activity, and thesequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 99%, 99.5%, 99.8%, ormore, or complete (100%) sequence identity to SEQ ID NO:21 over a regionof at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues,encodes at least one polypeptide having aldolase activity, and thesequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection.

In alternative aspects, the isolated, synthetic or recombinant nucleicacid encodes a polypeptide comprising a sequence as set forth in SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, or SEQ ID NO:30, or an enzymatically activefragment thereof. In one aspect these polypeptides have an aldolaseactivity.

In one aspect, the sequence comparison algorithm is a BLAST algorithm,such as a BLAST version 2.2.2 algorithm. In one aspect, the filteringsetting is set to blastall-p blastp-d “nr pataa”-F F and all otheroptions are set to default.

In one aspect, the aldolase activity comprises catalysis of theformation of a carbon-carbon bond. In one aspect, the aldolase activitycomprises an aldol condensation. The aldol condensation can have analdol donor substrate comprising an acetaldehyde and an aldol acceptorsubstrate comprising an aldehyde. The aldol condensation can yield aproduct of a single chirality. In one aspect, the aldolase activity isenantioselective. The aldolase activity can comprise a2-deoxyribose-5-phosphate aldolase (DERA) activity. The aldolaseactivity can comprise catalysis of the condensation of acetaldehyde asdonor and a 2(R)-hydroxy-3-(hydroxy or mercapto)-propionaldehydederivative to form a 2-deoxysugar. The aldolase activity can comprisecatalysis of the condensation of acetaldehyde as donor and a2-substituted acetaldehyde acceptor to form a 2,4,6-trideoxyhexose via a4-substituted-3-hydroxybutanal intermediate. The aldolase activity cancomprise catalysis of the generation of chiral aldehydes using twoacetaldehydes as substrates. The aldolase activity can comprisesenantioselective assembling of chiral β,δ-dihydroxyheptanoic acid sidechains. The aldolase activity can comprise enantioselective assemblingof the core of[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (Atorvastatin, or LIPITOR™), rosuvastatin (CRESTOR™) and/orfluvastatin (LESCOL™). The aldolase activity can comprise, with anoxidation step, synthesis of a3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone.

In one aspect, the isolated or recombinant nucleic acid encodes apolypeptide having an aldolase activity which is thermostable. Thepolypeptide can retain an aldolase activity under conditions comprisinga temperature anywhere in a range of between about 1° C. to about 5° C.,about 5° C. to about 15° C., about 15° C. to about 25° C., about 25° C.to about 37° C., 37° C. to about 95° C.; between about 55° C. to about85° C., between about 70° C. to about 95° C., or, between about 90° C.to about 95° C., 96° C., 97° C. or more. In another aspect, the isolatedor recombinant nucleic acid encodes a polypeptide having an aldolaseactivity which is thermotolerant. The polypeptide can retain an aldolaseactivity after exposure to a temperature anywhere in a range of betweenabout 1° C. to about 5° C., about 5° C. to about 15° C., about 15° C. toabout 25° C., about 25° C. to about 37° C., 37° C. to about 95° C.;between about 55° C. to about 85° C., between about 70° C. to about 95°C., or, between about 90° C. to about 95° C., 96° C., 97° C. or more.

In one aspect, the polypeptide can retain an aldolase activity underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4.In another aspect, the polypeptide can retain an aldolase activity underconditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5,pH 10, pH 10.5 or pH 1. In one aspect, the polypeptide can retain analdolase activity after exposure to conditions comprising about pH 6.5,pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In another aspect, the polypeptidecan retain an aldolase activity after exposure to conditions comprisingabout pH 7, pH 7.5 pH 8.0, pH 8.5, pH9,pH 9.5, pH 10,pH 10.5 or pH 11.

In one aspect, the isolated, synthetic or recombinant nucleic acidcomprises a sequence that hybridizes under stringent conditions to asequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, wherein the nucleic acid encodes a polypeptidehaving an aldolase activity. The nucleic acid can at least about 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850 or residues in length or the fulllength of the gene or transcript, with or without a signal sequence, asdescribed herein. The stringent conditions can be highly stringent,moderately stringent or of low stringency, as described herein. Thestringent conditions can include a wash step, e.g., a wash stepcomprising a wash in 0.2×SSC at a temperature of about 65° C. for about15 minutes.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide with an aldolase activity, wherein the probecomprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, ormore, consecutive bases of a sequence of the invention, e.g., asexemplary sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, and the probe identifies the nucleic acid by binding orhybridization. The probe can comprise an oligonucleotide comprising atleast about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, orabout 60 to 100 consecutive bases of a sequence as set forth in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide with an aldolase activity, wherein the probecomprises a nucleic acid of the invention, e.g., a nucleic acid havingat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, over a region of at least about 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850 or more consecutive residues, wherein thesequence identities are determined by analysis with a sequencecomparison algorithm or by visual inspection.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having an aldolaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. In one aspect, one or each member of theamplification primer sequence pair comprises an oligonucleotidecomprising at least about 10 to 50 consecutive bases of the sequence, orabout 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25consecutive bases of the sequence.

The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more residues of a nucleic acid of theinvention, and a second member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more residues of the complementary strand ofthe first member.

The invention provides aldolases generated by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. The invention provides methods of making an aldolase byamplification, e.g., polymerase chain reaction (PCR), using anamplification primer pair of the invention. In one aspect, theamplification primer pair amplifies a nucleic acid from a library, e.g.,a gene library, such as an environmental library.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having an aldolase activity comprising amplification of atemplate nucleic acid with an amplification primer sequence pair capableof amplifying a nucleic acid sequence of the invention, or fragments orsubsequences thereof. The amplification primer pair can be anamplification primer pair of the invention.

The invention provides expression cassettes comprising a nucleic acid ofthe invention. In one aspect, the expression cassette can comprise thenucleic acid that is operably linked to a promoter. The promoter can bea viral, bacterial, mammalian or plant promoter. In one aspect, theplant promoter can be a potato, rice, corn, wheat, tobacco or barleypromoter. The promoter can be a constitutive promoter. The constitutivepromoter can comprise CaMV35S. In another aspect, the promoter can be aninducible promoter. In one aspect, the promoter can be a tissue-specificpromoter or an environmentally regulated or a developmentally regulatedpromoter. Thus, the promoter can be, e.g., a seed-specific, aleaf-specific, a root-specific, a stem-specific or an abscission-inducedpromoter. In one aspect, the expression cassette can further comprise aplant or plant virus expression vector.

The invention provides cloning vehicles comprising an expressioncassette (e.g., a vector) of the invention or a nucleic acid of theinvention. The cloning vehicle can be a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome. The viral vector can comprise an adenovirus vector, aretroviral vector or an adeno-associated viral vector. The cloningvehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome(YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cell comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention,or a cloning vehicle of the invention. In one aspect, the transformedcell can be a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, an insect cell or a plant cell. In one aspect, the plant cell canbe a potato, wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention or an expression cassette (e.g., a vector) of theinvention. In one aspect, the animal is a mouse.

The invention provides transgenic plants comprising a nucleic acid ofthe invention or an expression cassette (e.g., a vector) of theinvention. The transgenic plant can be a corn plant, a potato plant, atomato plant, a wheat plant, an oilseed plant, a rapeseed plant, asoybean plant, a rice plant, a barley plant or a tobacco plant. Theinvention provides transgenic seeds comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention.The transgenic seed can be a corn seed, a wheat kernel, an oilseed, arapeseed (a canola plant), a soybean seed, a palm kernel, a sunflowerseed, a sesame seed, a peanut or a tobacco plant seed.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of an aldolase message in a cellcomprising administering to the cell or expressing in the cell anantisense oligonucleotide comprising a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions toa nucleic acid of the invention.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of an aldolase message in a cellcomprising administering to the cell or expressing in the cell anantisense oligonucleotide comprising a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions toa nucleic acid of the invention. The antisense oligonucleotide can bebetween about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80,about 60 to 100, about 70 to 110, or about 80 to 120 bases in length.

The invention provides methods of inhibiting the translation of analdolase, e.g., an aldolase, message in a cell comprising administeringto the cell or expressing in the cell an antisense oligonucleotidecomprising a nucleic acid sequence complementary to or capable ofhybridizing under stringent conditions to a nucleic acid of theinvention. The invention provides double-stranded inhibitory RNA (RNAi)molecules comprising a subsequence of a sequence of the invention. Inone aspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25or more duplex nucleotides in length. The invention provides methods ofinhibiting the expression of an aldolase, e.g., an aldolase, in a cellcomprising administering to the cell or expressing in the cell adouble-stranded inhibitory RNA (iRNA), wherein the RNA comprises asubsequence of a sequence of the invention.

The invention provides isolated or recombinant polypeptides comprising anucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ IDNO:6 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 150, 200, 250, 300 or more residues, wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by a visual inspection.

The invention provides isolated or recombinant polypeptides comprising anucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ IDNO:8 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 150, 200, 250, 300 or more residues, wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by a visual inspection.

The invention provides isolated or recombinant polypeptides comprising anucleic acid sequence having at least about 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQID NO:10 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250, 300 or more residues, wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by a visual inspection.

The invention provides isolated or recombinant polypeptides comprising anucleic acid sequence having at least about 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQID NO:12 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250, 300 or more residues, wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by a visual inspection.

The invention provides isolated or recombinant polypeptides comprising anucleic acid sequence having at least about 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity to SEQ ID NO:14 over a region of at least about 10, 15, 20, 25,30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300 or more residues,wherein the sequence identities are determined by analysis with asequence comparison algorithm or by a visual inspection.

The invention provides isolated or recombinant polypeptides comprising anucleic acid sequence having at least about 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to SEQ ID NO:16 over a region of atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250,300, or more residues, wherein the sequence identities are determined byanalysis with a sequence comparison algorithm or by a visual inspection.

The invention provides isolated or recombinant polypeptides comprising anucleic acid sequence having at least about 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to SEQ ID NO:18 over a region of at least about 10,15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300 or moreresidues, encodes at least one polypeptide having aldolase activity, andthe sequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection.

The invention provides isolated or recombinant polypeptides comprising anucleic acid sequence having at least about 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to SEQ ID NO:20 over a regionof at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200,250, 300, 350 or more residues, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

The invention provides isolated or recombinant polypeptides comprising anucleic acid sequence having at least about 99%, 99.5%, 99.8%, or more,or complete (100%) sequence identity to SEQ BD NO:22 over a region of atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250,300, 350 or more residues, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

The invention provides isolated or recombinant polypeptides encoded bynucleic acid comprising a sequence as set forth in SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29. In alternative aspects, theisolated, synthetic or recombinant polypeptides comprise a sequence asset forth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, or an enzymaticallyactive fragment thereof. In one aspect these polypeptides have analdolase activity.

Another aspect of the invention provides an isolated or recombinantpolypeptide or peptide including at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more consecutivebases of a polypeptide or peptide sequence of the invention (e.g., theexemplary SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ BD NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30), sequencessubstantially identical thereto, and the sequences complementarythereto. The peptide can be, e.g., an immunogenic fragment, a motif(e.g., a binding site), a signal sequence, a prepro sequence or anactive site.

In one aspect, the isolated or recombinant polypeptide of the invention(with or without a signal sequence) has an aldolase activity. In oneaspect, the aldolase activity comprises catalysis of the formation of acarbon-carbon bond. In one aspect, the aldolase activity comprises analdol condensation. The aldol condensation can have an aldol donorsubstrate comprising an acetaldehyde and an aldol acceptor substratecomprising an aldehyde. The aldol condensation can yield a product of asingle chirality. In one aspect, the aldolase activity isenantioselective. The aldolase activity can comprise a2-deoxyribose-5-phosphate aldolase (DERA) activity. The aldolaseactivity can comprise catalysis of the condensation of acetaldehyde asdonor and a 2(R)-hydroxy-3-(hydroxy or mercapto)-propionaldehydederivative to form a 2-deoxysugar. The aldolase activity can comprisecatalysis of the condensation of acetaldehyde as donor and a2-substituted acetaldehyde acceptor to form a 2,4,6-trideoxyhexose via a4-substituted-3-hydroxybutanal intermediate. The aldolase activity cancomprise catalysis of the generation of chiral aldehydes using twoacetaldehydes as substrates. The aldolase activity can comprisesenantioselective assembling of chiral β,δ-dihydroxyheptanoic acid sidechains. The aldolase activity can comprise enantioselective assemblingof the core of[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, or LIPITOR™), rosuvastatin (CRESTOR™) and/orfluvastatin (LESCOL™). The aldolase activity can comprise, with anoxidation step, synthesis of a3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone.

In one aspect, the aldolase activity is thermostable. A polypeptide ofthe invention can retain an aldolase activity under conditionscomprising a temperature anywhere in a range of between about 1° C. toabout 5° C., about 5° C. to about 15° C., about 15° C. to about 25° C.,about 25° C. to about 37° C., 37° C. to about 95° C.; between about 55°C. to about 85° C., between about 70° C. to about 95° C., or, betweenabout 90° C. to about 95° C., 96° C., 97° C. or more. In another aspect,the aldolase activity is thermotolerant. A polypeptide of the inventioncan retain an aldolase activity after exposure to a temperature anywherein a range of between about 1° C. to about 5° C., about 5° C. to about15° C., about 15° C. to about 25° C., about 25° C. to about 37° C., 37°C. to about 95° C.; between about 55° C. to about 85° C., between about70° C. to about 95° C., or, between about 90° C. to about 95° C., 96°C., 97° C. or more.

In one aspect, the polypeptide can retain an aldolase activity underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4.In another aspect, the polypeptide can retain an aldolase activity underconditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5,pH 10, pH 10.5 or pH 11. In one aspect, the polypeptide can retain analdolase activity after exposure to conditions comprising about pH 6.5,pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In another aspect, the polypeptidecan retain an aldolase activity after exposure to conditions comprisingabout pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH11.

In one aspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention that lacks a signal sequence and/or aprepro domain. In one aspect, the isolated or recombinant polypeptidecan comprise the polypeptide of the invention comprising a heterologoussignal sequence and/or prepro domain, such as a heterologous aldolase ora non-aldolase signal sequence.

In one aspect, the invention provides a signal sequence comprising apeptide comprising/consisting of a sequence as set forth in residues 1to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44 of a polypeptide ofthe invention, e.g., the exemplary SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30.

The invention provides isolated or recombinant peptides comprising anamino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or more, orcomplete sequence identity to residues 1 to 22 of SEQ ID NO:18, whereinthe sequence identities are determined by analysis with a sequencecomparison algorithm or by visual inspection. These peptides can act assignal sequences on its endogenous aldolase, on another aldolase, or aheterologous protein (a non-aldolase enzyme or other protein). In oneaspect, the invention provides chimeric proteins comprising a firstdomain comprising a signal sequence of the invention and at least asecond domain. The protein can be a fusion protein. The second domaincan comprise an enzyme. The enzyme can be an aldolase.

The invention provides chimeric polypeptides comprising at least a firstdomain comprising signal peptide (SP), a prepro domain, a catalyticdomain (CD), or an active site of an aldolase of the invention and atleast a second domain comprising a heterologous polypeptide or peptide,wherein the heterologous polypeptide or peptide is not naturallyassociated with the signal peptide (SP), prepro domain or catalyticdomain (CD). In one aspect, the heterologous polypeptide or peptide isnot an aldolase. The heterologous polypeptide or peptide can be aminoterminal to, carboxy terminal to or on both ends of the signal peptide(SP), prepro domain or catalytic domain (CD).

In one aspect, the aldolase activity comprises a specific activity atabout 37° C. in the range from about 1 to about 1200 units per milligram(U/mg) of protein, or, about 100 to about 1000 units per milligram ofprotein, or, about 200 to about 800 units per milligram of protein. Inanother aspect, the aldolase activity comprises a specific activity fromabout 100 to about 1000 units per milligram of protein, or, from about500 to about 750 units per milligram of protein. Alternatively, thealdolase activity comprises a specific activity at 37° C. in the rangefrom about 1 to about 750 units per milligram of protein, or, from about500 to about 1200 units per milligram of protein. In one aspect, thealdolase activity comprises a specific activity at 37° C. in the rangefrom about 1 to about 500 units per milligram of protein, or, from about750 to about 1000 units per milligram of protein. In another aspect, thealdolase activity comprises a specific activity at 37° C. in the rangefrom about 1 to about 250 units per milligram of protein. Alternatively,the aldolase activity comprises a specific activity at 37° C. in therange from about 1 to about 100 units per milligram of protein. Inanother aspect, the thermotolerance comprises retention of at least halfof the specific activity of the aldolase at 37° C. after being heated tothe elevated temperature. Alternatively, the thermotolerance cancomprise retention of specific activity at 37° C. in the range fromabout 1 to about 1200 units per milligram of protein, or, from about 500to about 1000 units per milligram of protein, after being heated to theelevated temperature. In another aspect, the thermotolerance cancomprise retention of specific activity at 37° C. in the range fromabout 1 to about 500 units per milligram of protein after being heatedto the elevated temperature.

The invention provides the isolated or recombinant polypeptide of theinvention, wherein the polypeptide comprises at least one glycosylationsite. In one aspect, glycosylation can be an N-linked glycosylation. Inone aspect, the polypeptide can be glycosylated after being expressed ina P. pastoris or a S. pombe.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, asolid or a gel.

The invention provides heterodimers comprising a polypeptide of theinvention and a second protein or domain. The second member of theheterodimer can be a different aldolase, a different enzyme or anotherprotein. In one aspect, the second domain can be a polypeptide and theheterodimer can be a fusion protein. In one aspect, the second domaincan be an epitope or a tag. In one aspect, the invention provideshomodimers comprising a polypeptide of the invention.

The invention provides immobilized polypeptides having an aldolaseactivity, wherein the polypeptide comprises a polypeptide of theinvention, a polypeptide encoded by a nucleic acid of the invention, ora polypeptide comprising a polypeptide of the invention and a seconddomain. In one aspect, the polypeptide can be immobilized on a cell, ametal, a resin, a polymer, a ceramic, a glass, a microelectrode, agraphitic particle, a bead, a gel, a plate, an array or a capillarytube.

The invention provides arrays comprising an immobilized polypeptide,wherein the polypeptide is an aldolase of the invention or is apolypeptide encoded by a nucleic acid of the invention. The inventionprovides arrays comprising an immobilized nucleic acid of the invention.The invention provides an array comprising an immobilized antibody ofthe invention.

The invention provides isolated or recombinant antibodies thatspecifically bind to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention. The antibody can be amonoclonal or a polyclonal antibody. The invention provides hybridomascomprising an antibody of the invention.

The invention provides methods of isolating or identifying a polypeptidewith an aldolase activity comprising the steps of: (a) providing anantibody of the invention; (b) providing a sample comprisingpolypeptides; and, (c) contacting the sample of step (b) with theantibody of step (a) under conditions wherein the antibody canspecifically bind to the polypeptide, thereby isolating or identifyingan aldolase. The invention provides methods of making an anti-aldolaseantibody comprising administering to a non-human animal a nucleic acidof the invention, or a polypeptide of the invention, in an amountsufficient to generate a humoral immune response, thereby making ananti-aldolase antibody.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid of the inventionoperably linked to a promoter; and, (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide. The method can furthercomprise transforming a host cell with the nucleic acid of step (a)followed by expressing the nucleic acid of step (a), thereby producing arecombinant polypeptide in a transformed cell. The method can furthercomprise inserting into a host non-human animal the nucleic acid of step(a) followed by expressing the nucleic acid of step (a), therebyproducing a recombinant polypeptide in the host non-human animal.

The invention provides methods for identifying a polypeptide having analdolase activity comprising the following steps: (a) providing apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention, or a fragment or variant thereof, (b) providing analdolase substrate; and, (c) contacting the polypeptide or a fragment orvariant thereof of step (a) with the substrate of step (b) and detectingan increase in the amount of substrate or a decrease in the amount ofreaction product, wherein a decrease in the amount of the substrate oran increase in the amount of the reaction product detects a polypeptidehaving an aldolase activity.

The invention provides methods for identifying an aldolase substratecomprising the following steps: (a) providing a polypeptide of theinvention or a polypeptide encoded by a nucleic acid of the invention;(b) providing a test substrate; and, (c) contacting the polypeptide ofstep (a) with the test substrate of step (b) and detecting an increasein the amount of substrate or a decrease in the amount of reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of the reaction product identifies the testsubstrate as an aldolase substrate.

The invention provides methods of determining whether a compoundspecifically binds to an aldolase comprising the following steps: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid and vector comprise a nucleic acidor vector of the invention; or, providing a polypeptide of the invention(b) contacting the polypeptide with the test compound; and, (c)determining whether the test compound specifically binds to thepolypeptide, thereby determining that the compound specifically binds tothe aldolase.

The invention provides methods for identifying a modulator of analdolase activity comprising the following steps: (a) providing apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention; (b) providing a test compound; (c) contacting thepolypeptide of step (a) with the test compound of step (b); and,measuring an activity of the aldolase, wherein a change in the aldolaseactivity measured in the presence of the test compound compared to theactivity in the absence of the test compound provides a determinationthat the test compound modulates the aldolase activity.

In one aspect, the aldolase activity is measured by providing analdolase substrate and detecting an increase in the amount of thesubstrate or a decrease in the amount of a reaction product. Thedecrease in the amount of the substrate or the increase in the amount ofthe reaction product with the test compound as compared to the amount ofsubstrate or reaction product without the test compound identifies thetest compound as an activator of aldolase activity. The increase in theamount of the substrate or the decrease in the amount of the reactionproduct with the test compound as compared to the amount of substrate orreaction product without the test compound identifies the test compoundas an inhibitor of aldolase activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence of the invention or a nucleic acid sequence ofthe invention.

In one aspect, the computer system can further comprise a sequencecomparison algorithm and a data storage device having at least onereference sequence stored thereon. The sequence comparison algorithm cancomprise a computer program that indicates polymorphisms. The computersystem can further comprising an identifier that identifies one or morefeatures in said sequence.

The invention provides computer readable mediums having stored thereon asequence comprising a polypeptide sequence of the invention or a nucleicacid sequence of the invention.

The invention provides methods for identifying a feature in a sequencecomprising the steps of: (a) reading the sequence using a computerprogram which identifies one or more features in a sequence, wherein thesequence comprises a polypeptide sequence of the invention or a nucleicacid sequence of the invention; and, (b) identifying one or morefeatures in the sequence with the computer program.

The invention provides methods for comparing a first sequence to asecond sequence comprising the steps of: (a) reading the first sequenceand the second sequence through use of a computer program which comparessequences, wherein the first sequence comprises a polypeptide sequenceof the invention or a nucleic acid sequence of the invention; and, (b)determining differences between the first sequence and the secondsequence with the computer program. In one aspect, the step ofdetermining differences between the first sequence and the secondsequence further comprises the step of identifying polymorphisms. In oneaspect, the method further comprises an identifier (and use of theidentifier) that identifies one or more features in a sequence. In oneaspect, the method comprises reading the first sequence using a computerprogram and identifying one or more features in the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide with an aldolase activity from anenvironmental sample comprising the steps of: (a) providing anamplification primer sequence pair for amplifying a nucleic acidencoding a polypeptide with an aldolase activity, wherein the primerpair is capable of amplifying a nucleic acid of the invention; (b)isolating a nucleic acid from the environmental sample or treating theenvironmental sample such that nucleic acid in the sample is accessiblefor hybridization to the amplification primer pair; and, (c) combiningthe nucleic acid of step (b) with the amplification primer pair of step(a) and amplifying nucleic acid from the environmental sample, therebyisolating or recovering a nucleic acid encoding a polypeptide with analdolase activity from an environmental sample. In one aspect, eachmember of the amplification primer sequence pair comprises anoligonucleotide comprising at least about 10 to 50 consecutive bases ofa nucleic acid sequence of the invention. In one aspect, theamplification primer sequence pair is an amplification pair of theinvention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide with an aldolase activity from anenvironmental sample comprising the steps of: (a) providing apolynucleotide probe comprising a nucleic acid sequence of theinvention; (b) isolating a nucleic acid from the environmental sample ortreating the environmental sample such that nucleic acid in the sampleis accessible for hybridization to a polynucleotide probe of step (a);(c) combining the isolated nucleic acid or the treated environmentalsample of step (b) with the polynucleotide probe of step (a); and, (d)isolating a nucleic acid that specifically hybridizes with thepolynucleotide probe of step (a), thereby isolating or recovering anucleic acid encoding a polypeptide with an aldolase activity from theenvironmental sample. In alternative aspects, the environmental samplecomprises a water sample, a liquid sample, a soil sample, an air sampleor a biological sample. In alternative aspects, the biological sample isderived from a bacterial cell, a protozoan cell, an insect cell, a yeastcell, a plant cell, a fungal cell or a mammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding an aldolase comprising the steps of: (a) providing a templatenucleic acid comprising a nucleic acid of the invention; (b) modifying,deleting or adding one or more nucleotides in the template sequence, ora combination thereof, to generate a variant of the template nucleicacid.

In one aspect, the method further comprises expressing the variantnucleic acid to generate a variant aldolase polypeptide. In alternativeaspects, the modifications, additions or deletions are introduced byerror-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, gene sitesaturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and/ora combination thereof. In alternative aspects, the modifications,additions or deletions are introduced by a method selected from thegroup consisting of recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and/or a combination thereof.

In one aspect, the method is iteratively repeated until an aldolasehaving an altered or different activity or an altered or differentstability from that of an aldolase encoded by the template nucleic acidis produced. In one aspect, the altered or different activity is analdolase activity under an acidic condition, wherein the aldolaseencoded by the template nucleic acid is not active under the acidiccondition. In one aspect, the altered or different activity is analdolase activity under a high temperature, wherein the aldolase encodedby the template nucleic acid is not active under the high temperature.In one aspect, the method is iteratively repeated until an aldolasecoding sequence having an altered codon usage from that of the templatenucleic acid is produced. The method can be iteratively repeated untilan aldolase gene having higher or lower level of message expression orstability from that of the template nucleic acid is produced.

The invention provides methods for modifying codons in a nucleic acidencoding an aldolase to increase its expression in a host cell, themethod comprising (a) providing a nucleic acid of the invention encodingan aldolase; and, (b) identifying a non-preferred or a less preferredcodon in the nucleic acid of step (a) and replacing it with a preferredor neutrally used codon encoding the same amino acid as the replacedcodon, wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding an aldolase, the method comprising (a) providing a nucleic acidof the invention encoding an aldolase; and, (b) identifying a codon inthe nucleic acid of step (a) and replacing it with a different codonencoding the same amino acid as the replaced codon, thereby modifyingcodons in a nucleic acid encoding an aldolase.

The invention provides methods for modifying codons in a nucleic acidencoding an aldolase to increase its expression in a host cell, themethod comprising (a) providing a nucleic acid of the invention encodingan aldolase; and, (b) identifying a non-preferred or a less preferredcodon in the nucleic acid of step (a) and replacing it with a preferredor neutrally used codon encoding the same amino acid as the replacedcodon, wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding an aldolase to decrease its expression in a host cell, themethod comprising (a) providing a nucleic acid of the invention encodingan aldolase; and, (b) identifying at least one preferred codon in thenucleic acid of step (a) and replacing it with a non-preferred or lesspreferred codon encoding the same amino acid as the replaced codon,wherein a preferred codon is a codon over-represented in codingsequences in genes in a host cell and a non-preferred or less preferredcodon is a codon under-represented in coding sequences in genes in thehost cell, thereby modifying the nucleic acid to decrease its expressionin a host cell. In alternative aspects, the host cell is a bacterialcell, a fungal cell, an insect cell, a yeast cell, a plant cell or amammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified aldolase active sites or substratebinding sites, wherein the modified active sites or substrate bindingsites are derived from a first nucleic acid comprising a sequenceencoding a first active site or a first substrate binding site themethod comprising: (a) providing a first nucleic acid encoding a firstactive site or first substrate binding site, wherein the first nucleicacid sequence comprises a nucleic acid of the invention; (b) providing aset of mutagenic oligonucleotides that encode naturally-occurring aminoacid variants at a plurality of targeted codons in the first nucleicacid; and, (c) using the set of mutagenic oligonucleotides to generate aset of active site-encoding or substrate binding site-encoding variantnucleic acids encoding a range of amino acid variations at each aminoacid codon that was mutagenized, thereby producing a library of nucleicacids encoding a plurality of modified aldolase active sites orsubstrate binding sites. In alternative aspects, the method comprisesmutagenizing the first nucleic acid of step (a) by a method comprisingan optimized directed evolution system, gene site-saturation mutagenesis(GSSM), and synthetic ligation reassembly (SLR). The method can furthercomprise mutagenizing the first nucleic acid of step (a) or variants bya method comprising error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR)and a combination thereof. The method can further comprise mutagenizingthe first nucleic acid of step (a) or variants by a method comprisingrecombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation and acombination thereof.

The invention provides methods for making a small molecule comprisingthe steps of: (a) providing a plurality of biosynthetic enzymes capableof synthesizing or modifying a small molecule, wherein one of theenzymes comprises an aldolase enzyme encoded by a nucleic acid of theinvention; (b) providing a substrate for at least one of the enzymes ofstep (a); and, (c) reacting the substrate of step (b) with the enzymesunder conditions that facilitate a plurality of biocatalytic reactionsto generate a small molecule by a series of biocatalytic reactions.

The invention provides methods for modifying a small molecule comprisingthe steps: (a) providing an aldolase enzyme encoded by a nucleic acid ofthe invention; (b) providing a small molecule; and, (c) reacting theenzyme of step (a) with the small molecule of step (b) under conditionsthat facilitate an enzymatic reaction catalyzed by the aldolase enzyme,thereby modifying a small molecule by an aldolase enzymatic reaction. Inone aspect, the method comprises providing a plurality of small moleculesubstrates for the enzyme of step (a), thereby generating a library ofmodified small molecules produced by at least one enzymatic reactioncatalyzed by the aldolase enzyme. In one aspect, the method furthercomprises a plurality of additional enzymes under conditions thatfacilitate a plurality of biocatalytic reactions by the enzymes to forma library of modified small molecules produced by the plurality ofenzymatic reactions. In one aspect, the method further comprises thestep of testing the library to determine if a particular modified smallmolecule that exhibits a desired activity is present within the library.The step of testing the library can further comprises the steps ofsystematically eliminating all but one of the biocatalytic reactionsused to produce a portion of the plurality of the modified smallmolecules within the library by testing the portion of the modifiedsmall molecule for the presence or absence of the particular modifiedsmall molecule with a desired activity, and identifying at least onespecific biocatalytic reaction that produces the particular modifiedsmall molecule of desired activity.

The invention provides methods for determining a functional fragment ofan aldolase enzyme comprising the steps of: (a) providing an aldolaseenzyme comprising an amino acid sequence of the invention; and, (b)deleting a plurality of amino acid residues from the sequence of step(a) and testing the remaining subsequence for an aldolase activity,thereby determining a functional fragment of an aldolase enzyme. In oneaspect, the aldolase activity is measured by providing an aldolasesubstrate and detecting an increase in the amount of the substrate or adecrease in the amount of a reaction product. In one aspect, a decreasein the amount of an enzyme substrate or an increase in the amount of thereaction product with the test compound as compared to the amount ofsubstrate or reaction product without the test compound identifies thetest compound as an activator of aldolase activity.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising the following steps: (a) making a modified cell bymodifying the genetic composition of a cell, wherein the geneticcomposition is modified by addition to the cell of a nucleic acid of theinvention; (b) culturing the modified cell to generate a plurality ofmodified cells; (c) measuring at least one metabolic parameter of thecell by monitoring the cell culture of step (b) in real time; and, (d)analyzing the data of step (c) to determine if the measured parameterdiffers from a comparable measurement in an unmodified cell undersimilar conditions, thereby identifying an engineered phenotype in thecell using real-time metabolic flux analysis. In one aspect, the geneticcomposition of the cell can be modified by a method comprising deletionof a sequence or modification of a sequence in the cell, or, knockingout the expression of a gene. In one aspect, the method can furthercomprise selecting a cell comprising a newly engineered phenotype. Inanother aspect, the method can comprise culturing the selected cell,thereby generating a new cell strain comprising a newly engineeredphenotype.

The invention provides methods of increasing thermotolerance orthermostability of an aldolase polypeptide, the method comprisingglycosylating an aldolase polypeptide, wherein the polypeptide comprisesat least thirty contiguous amino acids of a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid sequence of theinvention, thereby increasing the thermotolerance or thermostability ofthe aldolase polypeptide. In one aspect, the aldolase specific activitycan be thermostable or thermotolerant at a temperature in the range fromgreater than about 37° C. to about 95° C.

The invention provides methods for overexpressing a recombinant aldolasepolypeptide in a cell comprising expressing a vector comprising anucleic acid comprising a nucleic acid of the invention or a nucleicacid sequence of the invention, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by visualinspection, wherein overexpression is effected by use of a high activitypromoter, a dicistronic vector or by gene amplification of the vector.

The invention provides methods of making a transgenic plant comprisingthe following steps: (a) introducing a heterologous nucleic acidsequence into the cell, wherein the heterologous nucleic sequencecomprises a nucleic acid sequence of the invention, thereby producing atransformed plant cell; and (b) producing a transgenic plant from thetransformed cell. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a nucleic acid of the invention; (b) growing the plant underconditions wherein the heterologous nucleic acids sequence is expressedin the plant cell. The invention provides methods of expressing aheterologous nucleic acid sequence in a plant cell comprising thefollowing steps: (a) transforming the plant cell with a heterologousnucleic acid sequence operably linked to a promoter, wherein theheterologous nucleic sequence comprises a sequence of the invention; (b)growing the plant under conditions wherein the heterologous nucleicacids sequence is expressed in the plant cell.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one aspect of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence.

FIG. 5 illustrates the chemical formula of[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid, or, atorvastatin (LIPITOR™), as described in detail, below.

FIG. 6 is a schematic representation of an aldol reaction catalyzed byan aldolase, as described in detail, below.

FIG. 7 is a schematic representation of a DERA-catalyzed aldol synthesisof a side chain intermediate used, e.g., in the production of[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, or LIPITOR™) or rosuvastatin (CRESTOR™), asdescribed in detail, below.

FIG. 8 illustrates exemplary schemes for DERA-catalyzed production ofatorvastatin (LIPITOR™) or rosuvastatin (CRESTOR™) side chains, asdescribed in detail, below.

FIG. 9 is a schematic representation of an exemplary oxidation (abromine/barium carbonate oxidation) of the DERA-catalyzed intermediateto a chloro lactone, as described in detail, below.

FIG. 10 illustrates exemplary synthesis options starting from thelactone VI to the atorvastatin (LIPITOR™) or rosuvastatin (CRESTOR™)side chain, as described in detail, below.

FIG. 11 is a schematic representation of an exemplary complete[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, LIPITOR™) synthesis of the invention, with theintact lactone intermediate, as described in detail, below.

FIG. 12 illustrates the exemplary Route II DERA-catalyzed reaction ofthe invention, as described in detail, below.

FIG. 13 is a schematic representation of the exemplary Route IIIDERA-catalyzed reaction of the invention, as described in detail, below.

FIG. 14 illustrates exemplary syntheses using an aldolase, includingDERA-catalyzed syntheses of advanced statin intermediates that can beused, e.g., in the synthesis of atorvastatin (LIPITOR™) or rosuvastatin(CRESTOR™), as described in detail, below.

FIG. 15 illustrates an exemplary method of the invention comprisingoxidation of crude chlorolactol to crystalline chlorolactone with sodiumhypochlorite, as described in detail, below.

FIG. 16 illustrates an exemplary single step process for converting thelactone 3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone to either(3R,5S)-3,5,6-trihydroxyhexanoic acid or(3R,5R)-6-cyano-3,5,-dihydroxyhexanoic acid, as described in detail,below.

FIG. 17 illustrates an exemplary process for making6-chloro-2,4,6-trideoxyerythro-hexonolactone, and, rosuvastatin(CRESTOR™) and fluvastatin (LESCOL™), and their various intermediates,using a DERA.

FIG. 18 illustrates an exemplary process for making6-chloro-2,4,6-trideoxyerythro-hexonolactone,[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, LIPITOR™), and their various intermediates, using aDERA.

FIG. 19 illustrates the structure of rosuvastatin (CRESTOR™).

FIG. 20 illustrates the structure of fluvastatin (LESCOL™).

FIG. 21 illustrates exemplary process for the synthesis of statinintermediates and atorvastatin (LIPITOR™), rosuvastatin (CRESTOR™),fluvastatin (LESCOL™) and related compounds.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The present invention provides chemoenzymatic methods for the synthesisof chiral β,δ-dihydroxyheptanoic acid side chains,[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, LIPITOR™) (FIG. 5), rosuvastatin (CRESTOR™) (FIG.19), fluvastatin (LESCOL™) (FIG. 20), related compounds and theirintermediates.

The invention also provides intermediates of atorvastatin, rosuvastatinand related compounds having a chiral β,δ-dihydroxyheptanoic acid sidechain, and methods of making them.

The chemoenzymatic methods of the invention can use any polypeptidehaving an aldolase activity (e.g., an enzyme, a catalytic antibody),e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,including a polypeptide of the invention having an aldolase activity,e.g., the exemplary SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, or, a polypeptide encoded by a nucleic acid as set forth in SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29.

The invention provides enantioselective syntheses of various compoundsby using an aldolase in enzymatic aldol condensation. The aldolase canbe any aldolase, or, an aldolase of the invention (e.g., the exemplarySEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, or SEQ ID NO:30).

The polypeptides of the invention can have any aldolase or lyaseactivity. The enzymes of the invention can have the activity of anyaldolase, which are part of a large group of enzymes called lyases andare present in all organisms. The function of aldolases in vivo is oftenrelated to the degradative cleavage of metabolites. For example, thealdolases of the invention can catalyze C—C bond formation, and, in oneaspect, in a highly stereoselective way. As another example, apolypeptide of the invention can have a 2-deoxyribose-5-phosphatealdolase (DERA) activity, which can comprise catalysis of the reversiblealdol reaction between acetaldehyde and D-glyceraldehyde-3-phosphate togenerate D-2-deoxyribose-5-phosphate. DERA aldolase activity of theinvention can catalyze the reversible asymmetric aldol addition reactionof two aldehydes. Further exemplary activities of polypeptides of theinvention are described, below.

One aspect of the invention uses a 2-deoxyribose-5-phosphate aldolase(DERA) in a process to prepare a chiral β,δ-dihydroxyheptanoic acid sidechain. A DERA of the invention can assemble a statin side chain, e.g.,an atorvastatin (LIPITOR™) and/or a rosuvastatin (CRESTOR™) side chainand/or a fluvastatin (LESCOL™) side chain, including the setting of oneor both stereogenic centers; which, in one aspect, can be in a singletransformation. Low-cost starting materials can be used. As noted above,any polypeptide having an aldolase activity (e.g., an enzyme, acatalytic antibody, as, e.g., described in U.S. Pat. No. 6,368,839),e.g., including the exemplary aldolases of the invention SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, or SEQ ID NO:30, or enzymatically active fragments thereof, canbe used.

One aspect of this invention provides a DERA-catalyzed synthesis of anintermediate of formula II from an acetaldehyde and the aldehyde offormula III, as illustrated in FIG. 7. In intermediate II and aldehydeIII the R group can be a hydrogen, an alkyl group, an alkoxy group, ahalogen (e.g., a chlorine) or an azido group.

The term “alkoxy”, as used herein alone or as part of another group,denotes an alkyl group bonded through an oxygen linkage (—O—). The term“alkyl”, as used herein alone or as part of another group, denotesoptionally substituted, straight and branched chain saturatedhydrocarbon groups, in one aspect having 1 to 12 carbons in the normalchain. Exemplary unsubstituted such groups include methyl, ethyl,propyl, isopropyl, n-butyl, isobutyl, hexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, and the like. Exemplary substituents mayinclude the following groups: halo, alkoxy, alkylthio, alkenyl, alkynyl,aryl, cycloalkyl, cycloalkenyl, hydroxy or protected hydroxy, carboxyl,alkyloxycarbonyl, amino, mono- or dialkyloamino or thiol. The term“halogen” or “halo”, as used herein alone or as part of another group,denotes chlorine, bromine, iodine, and fluorine. The term azido, as usedherein alone or as part of another group, denotes an N₃ group. The termcyano, as used herein alone or as part of another group, denotes an —C≡Ngroup.

In one aspect, as illustrated in FIG. 7, the aldehyde of formula III isadmixed with acetaldehyde and DERA in an aqueous medium to form areaction mixture. In one aspect, the reaction mixture is maintained at aparticular pH value and temperature for a time period sufficient for theintermediate II to form and be recovered. In this reaction acetaldehydeis a donor substrate and the aldehyde III is the acceptor substrate. Inone aspect, the acetaldehyde is in stoichiometric excess over theacceptor. In one aspect, the ratio of donor to acceptor can be about1.5:1 to about 5:1 on a molar basis. In another aspect, the ratio ofdonor to acceptor can be about 2.5:1 to about 4:1. In one aspect, the pHvalue of the reaction mixture can be between about pH 6.5 and about pH8.5. In one aspect, the pH of the initially formed reaction mixturestays the same throughout the course of the reaction.

In one aspect, the reaction is carried out in the absence of light. Thecomponents can be admixed in the light, and the resulting reactionmixture can be shielded from the light. The process can be carried outin the absence of oxygen. In one aspect, the reaction can be carried outin an atmosphere of nitrogen, argon or a similar gas. See, for example,U.S. Pat. No. 5,795,749.

Alternative exemplary processes are illustrated in FIG. 8. Theintermediate of formula II can be converted through several chemicaltransformations to a lactone (IV), which in turn can serve as theintermediate for the production of Atorvastatin (see formula XI, FIG.11, and FIG. 1). Alternatively, a ring-open ester intermediate of theformula V (FIG. 8) can be prepared.

Route I (FIG. 8) exemplifies one aspect of this invention, whereinaldehyde is used as the donor and an aldehyde of formula III (FIG. 7 or8), wherein R is halogen (e.g., R is a chlorine, see aldehyde of formulaIII, FIG. 7), used as the aldehyde receptor in the aldolase enzymereaction. In one aspect, the aldehyde of formula III can bechloroacetaldehyde. An advantage of this exemplary approach may be thatboth starting aldehyde materials are low cost and readily available. Inone aspect, the invention provides a DERA enzyme catalyzing thisreaction with high efficiency.

In alternative aspects, the second step of the transformation in thisroute can involve oxidation of the intermediate of formula(Intermediate) II to a lactone IV (FIG. 8). See Routes I, II and III ofFIG. 8. In one aspect, this transformation can be performed on theunpurified crude product (intermediate of formula II) from theDERA-catalyzed reaction.

As illustrated in FIG. 8, in exemplary Route I, the synthesis of achloro lactone IV from halogenated intermediate II can comprise CN—displacement, lactal oxidation and nitrile reduction. In another aspect,in exemplary Route II, the synthesis of a cyano lactone IV from a cyanointermediate II can comprise lactal oxidation and nitrile reduction. Inanother aspect, in exemplary Route III, the synthesis of a nitrilelactone IV from a nitrile intermediate II can comprise lactal oxidationand azide reduction.

In one aspect, as illustrated in FIG. 9, the product6-chloro-2,4,6-trideoxyerythro-hexonolactone (chloro-lactone VI) iscrystalline and can be purified from the crude mixture byrecrystallization. In one aspect, this transformation can be carried outunder oxidation conditions comprising bromine (Br₂), BrCO₃ and water. Inone aspect, the oxidation conditions comprise sodium hypochlorite(NaOCl) in acetic acid (HOAc) and water

The 6-chloro-2,4,6-trideoxyerythro-hexonolactone (chloro-lactone VI) canbe converted to the final protected side chain intermediate in a numberof ways, as illustrated in FIG. 10, Routes A, B, and C.

One exemplary route entails cyanide displacement of chloride on thechlorinated lactone VI (FIG. 10, Route A). In one aspect, NaCN is usedfor cyanide displacement. This can be followed by ring opening or bycarrying the halogenated lactone IV through to the end of synthesis. Theadvantage of keeping the lactone IV intact is that it may obviate theneed for protection and deprotection steps. The ring can be opened bytreating the cyano lactone IX with MeOH/Dowex or MeOH/K₂CO₃ tosynthesize a cyano intermediate VII. Alternatively, the cyano lactone IX(FIG. 10, Route A) can be converted to an aminated (H₂N—) lactone IV.

Alternatively, as illustrated in Route B, FIG. 10, the cyanidedisplacement can be performed on an open-chain intermediate. The ringcan be opened by treating the lactone IX with MeOH/Dowex or MeOH/K₂CO₃resulting in the formation of a chlorinated intermediate VII. NaCN canbe used for cyanide displacement. The product is a cyano intermediateVII (which can be processed to intermediate VIII).

In another aspect, the ring opening can be achieved by treating thehalogenated lactone VI with MeOH/NaCN to obtain a cyano esterintermediate VII (Route C, FIG. 10). Route C can cut a step from theprocess by allowing the lactone opening and the cyanide displacement tooccur in one pot. The cyano ester intermediate VII can be processed tointermediate VIII. In one aspect, the process can utilize a tert-butylester rather than a methyl ester. If necessary, a transesterificationcan be performed to convert the methyl ester to the tert-butyl ester.

As illustrated in FIG. 11, the invention provides an alternativesynthetic route starting from an intact chloro lactone intermediate IV,wherein IV is converted to the intermediate X, which in turn isconverted to (R)-Ethyl-4-Cyano-3-Hydroxybutyrate, or, Atorvastatin(LIPITOR™) XI.

In FIG. 8, exemplary Route II occurs in one step by omitting the cyanidedisplacement step after the post-enzymatic process (see also FIG. 12).The aldehyde of formula III (wherein R is a cyano group) is notcommercially available. The same cyano-lactone (lactone IV) or cyanoopen chain intermediates (ester intermediate V) as above in FIG. 10) areaccessible from the cyano lactal intermediate II of FIG. 12.

In FIG. 8, exemplary route III also can occur one step by omitting thecyanide displacement step after the post-enzymatic process (see alsoFIG. 13). However, the starting material azido aldehyde (the aldehyde offormula III wherein R is N₃ has to be synthesized. From the azido lactalproduct (intermediate II), the same alternative routes are accessible asdescribed above, wherein both lactone IV and open-chain esterintermediate V can be developed.

The invention also provides a novel methodologies for the synthesis ofstatin intermediates using a deoxyribose-5-phosphate aldolase (DERA),which can be an enzyme of the invention. In one aspect, the inventionprovides for the conversion of Compound 1 of FIG. 14 to intermediatesfor the synthesis of statin intermediates, including atorvastatin(LIPITOR™) and rosuvastatin (CRESTOR™), by ring-opening and nucleophilicdisplacement with cyanide or hydroxide, respectively. The inventionprovides a process as set forth in FIG. 14. In one aspect, the methodcomprises use of NaCN (e.g., at 3 equivalents), DMF, and water (e.g., 5%H₂0). In one aspect, this reaction is run under conditions comprisingabout 40° C. and/or about 20 hours. In one aspect, the inventionprovides a process wherein the lactone3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone (Compound 1 of FIG.14) is made by a 2-step process integrating a biocatalytic step usingdeoxyribose-5-phosphate aldolase (DERA), e.g., an aldolase of theinvention, with a chemical oxidation step. The lactone3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone (compound 1 of FIG.14) can readily be converted into side-chain intermediates for thesynthesis of a variety of statin-type HMG-CoA reductase inhibitors,including atorvastatin (LIPITOR™), rosuvastatin (CRESTOR™), andfluvastatin (LESCOL™), see FIG. 14, FIG. 17 and FIG. 18.

In one aspect, the processes of the invention provide a significantimprovement in enzyme load and yield for DERAs by running a fed-batchreaction to gradually add the substrates acetaldehyde andchloroacetaldehyde to the enzyme.

A serious limitation to the DERA process as originally described in theliterature was the requirement of a high percentage of catalyst (enzymeload). For instance, to produce 10 grams of3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexose, about 2 grams of DERA wasrequired (20% enzyme load). The cause of the high enzyme requirement wasidentified to be inhibition by the substrate chloroacetaldehyde. Theinvention provides processes for overcoming this requirement by using afed-batch process. In one aspect, substrates are fed into the reactionover a several hour period, e.g., a 2 to 3 hour period (e.g., at roomtemperature) at a rate such that they are consumed as fast as they areadded, and chloroacetaldehyde does not reach inhibitory concentration.Under these conditions, enzyme load for E. coli DERA was reduced fromabout 20% to about 5%. This improvement also applies to any DERA,including the exemplary polypeptides of the invention, e.g., SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, or SEQ ID NO:30, or enzymatically active fragmentsthereof. In one aspect, the process is carried out in the range of 2 to4% enzyme load. Substrates are fed to a final concentration of about 600to 800 mM chloroacetaldehyde and about 1.2 to 1.6 M acetaldehyde. Thereaction can be run on a large scale, e.g., a 1-liter (or greater)scale, with isolation of 75 grams crude product.

The published procedure for oxidation of a lactol to a lactone, e.g.,the lactone 3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone(compound 1 of FIG. 14), uses bromine as the oxidant in the presence ofbarium carbonate and water. While this method is effective, the cost andtoxicity of bromine are issues for process scale. The invention providesa novel process wherein this oxidation can be performed in the sameyield with inexpensive sodium hypochlorite (bleach) in acetic acid, asillustrated in FIG. 15. In one aspect, the substrate is dissolved inglacial acetic acid at a concentration of 750 mM, and 1 equivalent ofaqueous sodium hypochlorite is fed into the solution over 3 hours, atroom temperature. 75 grams of crude lactol was converted to 40 grams ofpure 3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone (compound 1 ofFIG. 14) by this process. FIG. 15 illustrates the oxidation of crudechlorolactol to crystalline chlorolactone with sodium hypochlorite.

In one aspect, the lactone3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone (compound 1 of FIG.14, see also FIG. 17, FIG. 18) is converted in a single step to either(3R,5S)-3,5,6-trihydroxyhexanoic acid or(3R,5R)-6-cyano-3,5,-dihydroxyhexanoic acid (FIG. 16). The formercompound can be converted to rosuvastatin (CRESTOR™), fluvastatin(LESCOL™) and other statins, whereas the cyano compound can be convertedto atorvastatin (LIPITOR™). Both methods go through a commonintermediate, the epoxide (-(3R,5S-3-hydroxy-4-oxiranylbutyric acidsodium salt) shown in brackets in FIG. 16. See Example 2, below.

General Methods

The present invention provides novel biochemical processes for theproduction of chiral β,δ-dihydroxyheptanoic acid side chains, includingstatins, and compositions comprising these side chains, e.g.,[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, LIPITOR™), rosuvastatin (CRESTOR™), fluvastatin(LESCOL™) related compounds and various intermediates. The inventionalso provides novel aldolases, which, in one aspect, can be used topractice the methods of the invention. The skilled artisan willrecognize that the starting and intermediate compounds used in themethods of the invention can be synthesized using a variety ofprocedures and methodologies, which are well described in the scientificand patent literature, e.g., Organic Syntheses Collective Volumes,Gilman et al. (Eds) John Wiley & Sons, Inc., NY; Venuti (1989) PharmRes. 6:867-873. The invention can be practiced in conjunction with anymethod or protocol known in the art, which are well described in thescientific and patent literature. Enzymes of the invention, and theenzymes used in the methods of the invention, can be produced by anysynthetic or recombinant method, or, they may be isolated from a naturalsource, or, a combination thereof.

The nucleic acids and proteins of the invention can be detected,confirmed and quantified by any of a number of means well known to thoseof skill in the art. General methods for detecting both nucleic acidsand corresponding proteins include analytic biochemical methods such asspectrophotometry, radiography, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,and various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like. The detection of nucleic acidscan be by well known methods such as Southern analysis, northernanalysis, gel electrophoresis, PCR, radiolabeling, scintillationcounting, and affinity chromatography.

The discussion of the general methods given herein is intended forillustrative purposes only. Other alternative methods and embodimentswill be apparent to those of skill in the art upon review of thisdisclosure.

Generating and Manipulating Nucleic Acids

The invention provides isolated, synthetic or recombinant nucleic acids(e.g., the exemplary SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQID NO:29; or nucleic acids encoding the polypeptides of the invention,e.g., SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, or enzymaticallyactive fragments thereof). In one aspect, the nucleic acids encode apolypeptide having an aldolase activity.

Nucleic acids encoding aldolases of the invention, and enzymes used topractice the methods of the invention, whether RNA, cDNA, genomic DNA,vectors, viruses or hybrids thereof, may be isolated from a variety ofsources, genetically engineered, amplified, and/or expressed/generatedrecombinantly. Recombinant polypeptides generated from these nucleicacids can be individually isolated or cloned and tested for a desiredactivity. Any recombinant expression system can be used, includingbacterial, mammalian, yeast, insect or plant cell expression systems.Nucleic acids used to practice the methods of the invention, and to makethe polynucleotides and polypeptide of the invention, can be generatedusing amplification methods, which are also well known in the art, andinclude, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS,A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y.(1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y,ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560;Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario).

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440 3444; Frenkel (1995) Free Radic. Biol. Med. 19:373 380; Blommers(1994) Biochemistry 33:7886 7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y (1993). Anotheruseful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

Another useful means of obtaining and manipulating nucleic acids of theinvention, or nucleic acids used to practice the methods of theinvention, is to clone from genomic samples, and, if desired, screen andre-clone inserts isolated or amplified from, e.g., genomic clones orcDNA clones. Sources of nucleic acid used in the methods of theinvention include genomic or cDNA libraries contained in, e.g.,mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lacI promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding thealdolases of the invention. Expression vectors and cloning vehicles ofthe invention can comprise viral particles, baculovirus, phage,plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes,viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, Aspergillus and yeast).Vectors of the invention can include chromosomal, non-chromosomal andsynthetic DNA sequences. Large numbers of suitable vectors are known tothose of skill in the art, and are commercially available. Exemplaryvectors are include: bacterial: pQE vectors (Qiagen), pBluescriptplasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a,pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, anyother plasmid or other vector may be used so long as they are replicableand viable in the host. Low copy number or high copy number vectors maybe employed with the present invention.

The expression vector may comprise a promoter, a ribosome-binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A DNA sequence may be inserted into a vector by a variety of procedures.In general, the DNA sequence is ligated to the desired position in thevector following digestion of the insert and the vector with appropriaterestriction endonucleases. Alternatively, blunt ends in both the insertand the vector may be ligated. A variety of cloning techniques are knownin the art, e.g., as described in Ausubel and Sambrook. Such proceduresand others are deemed to be within the scope of those skilled in theart.

The vector may be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which may be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding an aldolase of theinvention, a vector of the invention. The host cell may be any of thehost cells familiar to those skilled in the art, including prokaryoticcells, eukaryotic cells, such as bacterial cells, fungal cells, yeastcells, mammalian cells, insect cells, or plant cells. Exemplarybacterial cells include E. coli, Streptomyces, Bacillus subtilis,Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus. Exemplary insect cellsinclude Drosophila S2 and Spodoptera Sf9. Exemplary animal cells includeCHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation; see, e.g., Davis, L.,Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts and other cell linescapable of expressing proteins from a compatible vector, such as theC127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids encoding the polypeptides ofthe invention, or modified nucleic acids, can be reproduced by, e.g.,amplification. The invention provides amplification primer sequencepairs for amplifying nucleic acids encoding polypeptides with analdolase activity. In one aspect, the primer pairs are capable ofamplifying nucleic acid sequences of the invention, e.g., including theexemplary SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, or subsequences thereof, nucleic acids encoding SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, or SEQ ID NO:30, or enzymatically active fragments thereof, orsubsequences thereof, etc. One of skill in the art can designamplification primer sequence pairs for any part of or the full lengthof these sequences.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having an aldolaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. In alternative aspects, one or each member of theamplification primer sequence pair can comprise an oligonucleotidecomprising at least about 10 to 50 consecutive bases of a sequence ofthe invention, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or more consecutive bases of a sequence ofthe invention. The invention provides amplification primer pairs,wherein the primer pair comprises a first member having a sequence asset forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues of a nucleicacid of the invention, and a second member having a sequence as setforth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues of thecomplementary strand of the first member. The invention providesaldolases generated by amplification, e.g., polymerase chain reaction(PCR), using an amplification primer pair of the invention. Theinvention provides methods of making an aldolase by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. In one aspect, the amplification primer pair amplifies anucleic acid from a library, e.g., a gene library, such as anenvironmental library.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified. The skilledartisan can select and design suitable oligonucleotide amplificationprimers. Amplification methods are also well known in the art, andinclude, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS,A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y.(1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y.,ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560;Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids comprising sequences having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to an exemplary nucleic acid of the invention(e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,and nucleic acids encoding SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, orenzymatically active fragments thereof) over a region of at least about50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550 or more, residues. The invention providespolypeptides comprising sequences having at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto an exemplary polypeptide of the invention. The extent of sequenceidentity (homology) may be determined using any computer program andassociated parameters, including those described herein, such as BLAST2.2.2. or FASTA version 3.0t78, with the default parameters.

In alternative embodiments, the sequence identify can be over a regionof at least about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350,400 consecutive residues, or the full length of the nucleic acid orpolypeptide. The extent of sequence identity (homology) may bedetermined using any computer program and associated parameters,including those described herein, such as BLAST 2.2.2. or FASTA version3.0t78, with the default parameters.

Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. It will beappreciated that the nucleic acid sequences as set forth herein can berepresented in the traditional single character format (see, e.g.,Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co., New York) orin any other format which records the identity of the nucleotides in asequence.

Various sequence comparison programs identified herein are used in thisaspect of the invention. Protein and/or nucleic acid sequence identities(homologies) may be evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are not limited to, TBLASTN, BLASTP, FASTA,TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higginset al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol.Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272,1993).

Homology or identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection. For sequencecomparison, one sequence can act as a reference sequence (an exemplarysequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, etc.) to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous residues. For example, inalternative aspects of the invention, continugous residues ranginganywhere from 20 to the full length of an exemplary sequence of theinvention are compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned. Ifthe reference sequence has the requisite sequence identity to anexemplary sequence of the invention, e.g., 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to a sequence of the invention, that(reference) sequence is within the scope of the invention. Inalternative embodiments, subsequences ranging from about 20 to 600,about 50 to 200, and about 100 to 150 are compared to a referencesequence of the same number of contiguous positions after the twosequences are optimally aligned. Methods of alignment of sequence forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of person & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project(Gibbs, 1995). Several genomes have been sequenced, e.g., M. genitalium(Fraser et al., 1995), M. jannaschii (Bult et al., 1996), H. influenzae(Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast(S. cerevisiae) (Mewes et al., 1997), and D. melanogaster (Adams et al.,2000). Significant progress has also been made in sequencing the genomesof model organism, such as mouse, C. elegans, and Arabadopsis sp.Databases containing genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the internet.

BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practicethe invention. They are described, e.g., in Altschul (1977) Nuc. AcidsRes. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The BLAST algorithm also performs a statisticalanalysis of the similarity between two sequences (see, e.g., Karlin &Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure ofsimilarity provided by BLAST algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a matchbetween two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered similar to a referencessequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, morepreferably less than about 0.01, and most preferably less than about0.001. In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”). Forexample, five specific BLAST programs can be used to perform thefollowing task: (1) BLASTP and BLAST3 compare an amino acid querysequence against a protein sequence database; (2) BLASTN compares anucleotide query sequence against a nucleotide sequence database; (3)BLASTX compares the six-frame conceptual translation products of a querynucleotide sequence (both strands) against a protein sequence database;(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and, (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database. The BLAST programs identify homologous sequences byidentifying similar segments, which are referred to herein as“high-scoring segment pairs,” between a query amino or nucleic acidsequence and a test sequence which is preferably obtained from a proteinor nucleic acid sequence database. High-scoring segment pairs arepreferably identified (i.e., aligned) by means of a scoring matrix, manyof which are known in the art. Preferably, the scoring matrix used isthe BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992;Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, thePAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978, Matrices for Detecting Distance Relationships:Atlas of Protein Sequence and Structure, Washington: National BiomedicalResearch Foundation).

In one aspect of the invention, to determine if a nucleic acid has therequisite sequence identity to be within the scope of the invention, theNCBI BLAST 2.2.2 programs is used default options to blastp. There areabout 38 setting options in the BLAST 2.2.2 program. In this exemplaryaspect of the invention, all default values are used except for thedefault filtering setting (i.e., all parameters set to default exceptfiltering which is set to OFF); in its place a “−F F” setting is used,which disables filtering. Use of default filtering often results inKarlin-Altschul violations due to short length of sequence.

The default values used in this exemplary aspect of the inventioninclude:

-   -   “Filter for low complexity: ON    -   Word Size: 3    -   Matrix: Blosum62    -   Gap Costs: Existence: 11        -   Extension: 1”

Other default settings are: filter for low complexity OFF, word size of3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and a gapextension penalty of −1.

An exemplary NCBI BLAST 2.2.2 program setting is set forth in Example 1,below. Note that the “−W” option defaults to 0. This means that, if notset, the word size defaults to 3 for proteins and 11 for nucleotides.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico the sequence of the invention can bestored, recorded, and manipulated on any medium which can be read andaccessed by a computer. Accordingly, the invention provides computers,computer systems, computer readable mediums, computer programs productsand the like recorded or stored thereon the nucleic acid and polypeptidesequences of the invention, e.g., an exemplary sequence of theinvention, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, etc. As used herein, the words “recorded” and “stored” refer to aprocess for storing information on a computer medium. A skilled artisancan readily adopt any known methods for recording information on acomputer readable medium to generate manufactures comprising one or moreof the nucleic acid and/or polypeptide sequences of the invention.

Another aspect of the invention is a computer readable medium havingrecorded thereon at least one nucleic acid and/or polypeptide sequenceof the invention. Computer readable media include magnetically readablemedia, optically readable media, electronically readable media andmagnetic/optical media. For example, the computer readable media may bea hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital VersatileDisk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) aswell as other types of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems, which store and manipulate the sequencesand sequence information described herein. One example of a computersystem 100 is illustrated in block diagram form in FIG. 1. As usedherein, “a computer system” refers to the hardware components, softwarecomponents, and data storage components used to analyze a nucleotide orpolypeptide sequence of the invention. The computer system 100 caninclude a processor for processing, accessing and manipulating thesequence data. The processor 105 can be any well-known type of centralprocessing unit, such as, for example, the Pentium III from IntelCorporation, or similar processor from Sun, Motorola, Compaq, AMD orInternational Business Machines. The computer system 100 is a generalpurpose system that comprises the processor 105 and one or more internaldata storage components 110 for storing data, and one or more dataretrieving devices for retrieving the data stored on the data storagecomponents. A skilled artisan can readily appreciate that any one of thecurrently available computer systems are suitable.

In one aspect, the computer system 100 includes a processor 105connected to a bus which is connected to a main memory 115 (preferablyimplemented as RAM) and one or more internal data storage devices 110,such as a hard drive and/or other computer readable media having datarecorded thereon. The computer system 100 can further include one ormore data retrieving device 118 for reading the data stored on theinternal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (e.g., via the internet)etc. In some embodiments, the internal data storage device 110 is aremovable computer readable medium such as a floppy disk, a compactdisk, a magnetic tape, etc. containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100. Software for accessing and processing the nucleotide oramino acid sequences of the invention can reside in main memory 115during execution.

In some aspects, the computer system 100 may further comprise a sequencecomparison algorithm for comparing a nucleic acid sequence of theinvention. The algorithm and sequence(s) can be stored on a computerreadable medium. A “sequence comparison algorithm” refers to one or moreprograms which are implemented (locally or remotely) on the computersystem 100 to compare a nucleotide sequence with other nucleotidesequences and/or compounds stored within a data storage means. Forexample, the sequence comparison algorithm may compare the nucleotidesequences of an exemplary sequence stored on a computer readable mediumto reference sequences stored on a computer readable medium to identifyhomologies or structural motifs.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user. FIG. 2 is a flow diagramillustrating one aspect of a process 200 for comparing a new nucleotideor protein sequence with a database of sequences in order to determinethe homology levels between the new sequence and the sequences in thedatabase. The database of sequences can be a private database storedwithin the computer system 100, or a public database such as GENBANKthat is available through the Internet. The process 200 begins at astart state 201 and then moves to a state 202 wherein the new sequenceto be compared is stored to a memory in a computer system 100. Asdiscussed above, the memory could be any type of memory, including RAMor an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200. If a determination is made that the two sequences are thesame, the process 200 moves to a state 214 wherein the name of thesequence from the database is displayed to the user. This state notifiesthe user that the sequence with the displayed name fulfills the homologyconstraints that were entered. Once the name of the stored sequence isdisplayed to the user, the process 200 moves to a decision state 218wherein a determination is made whether more sequences exist in thedatabase. If no more sequences exist in the database, then the process200 terminates at an end state 220. However, if more sequences do existin the database, then the process 200 moves to a state 224 wherein apointer is moved to the next sequence in the database so that it can becompared to the new sequence. In this manner, the new sequence isaligned and compared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.Accordingly, one aspect of the invention is a computer system comprisinga processor, a data storage device having stored thereon a nucleic acidsequence of the invention and a sequence comparer for conducting thecomparison. The sequence comparer may indicate a homology level betweenthe sequences compared or identify structural motifs, or it may identifystructural motifs in sequences which are compared to these nucleic acidcodes and polypeptide codes.

FIG. 3 is a flow diagram illustrating one embodiment of a process 250 ina computer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it can be a single letter amino acid code so thatthe first and sequence sequences can be easily compared. A determinationis then made at a decision state 264 whether the two characters are thesame. If they are the same, then the process 250 moves to a state 268wherein the next characters in the first and second sequences are read.A determination is then made whether the next characters are the same.If they are, then the process 250 continues this loop until twocharacters are not the same. If a determination is made that the nexttwo characters are not the same, the process 250 moves to a decisionstate 274 to determine whether there are any more characters eithersequence to read. If there are not any more characters to read, then theprocess 250 moves to a state 276 wherein the level of homology betweenthe first and second sequences is displayed to the user. The level ofhomology is determined by calculating the proportion of charactersbetween the sequences that were the same out of the total number ofsequences in the first sequence. Thus, if every character in a first 100nucleotide sequence aligned with a every character in a second sequence,the homology level would be 100%.

Alternatively, the computer program can compare a reference sequence toa sequence of the invention to determine whether the sequences differ atone or more positions. The program can record the length and identity ofinserted, deleted or substituted nucleotides or amino acid residues withrespect to the sequence of either the reference or the invention. Thecomputer program may be a program which determines whether a referencesequence contains a single nucleotide polymorphism (SNP) with respect toa sequence of the invention, or, whether a sequence of the inventioncomprises a SNP of a known sequence. Thus, in some aspects, the computerprogram is a program which identifies SNPs. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method can be performed by reading a sequenceof the invention and the reference sequences through the use of thecomputer program and identifying differences with the computer program.

In other aspects the computer based system comprises an identifier foridentifying features within a nucleic acid or polypeptide of theinvention. An “identifier” refers to one or more programs whichidentifies certain features within a nucleic acid sequence. For example,an identifier may comprise a program which identifies an open readingframe (ORF) in a nucleic acid sequence. FIG. 4 is a flow diagramillustrating one aspect of an identifier process 300 for detecting thepresence of a feature in a sequence. The process 300 begins at a startstate 302 and then moves to a state 304 wherein a first sequence that isto be checked for features is stored to a memory 115 in the computersystem 100. The process 300 then moves to a state 306 wherein a databaseof sequence features is opened. Such a database would include a list ofeach feature's attributes along with the name of the feature. Forexample, a feature name could be “Initiation Codon” and the attributewould be “ATG”. Another example would be the feature name “TAATAA Box”and the feature attribute would be “TAATAA”. An example of such adatabase is produced by the University of Wisconsin Genetics ComputerGroup. Alternatively, the features may be structural polypeptide motifssuch as alpha helices, beta sheets, or functional polypeptide motifssuch as enzymatic active sites, helix-turn-helix motifs or other motifsknown to those skilled in the art. Once the database of features isopened at the state 306, the process 300 moves to a state 308 whereinthe first feature is read from the database. A comparison of theattribute of the first feature with the first sequence is then made at astate 310. A determination is then made at a decision state 316 whetherthe attribute of the feature was found in the first sequence. If theattribute was found, then the process 300 moves to a state 318 whereinthe name of the found feature is displayed to the user. The process 300then moves to a decision state 320 wherein a determination is madewhether move features exist in the database. If no more features doexist, then the process 300 terminates at an end state 324. However, ifmore features do exist in the database, then the process 300 reads thenext sequence feature at a state 326 and loops back to the state 310wherein the attribute of the next feature is compared against the firstsequence. If the feature attribute is not found in the first sequence atthe decision state 316, the process 300 moves directly to the decisionstate 320 in order to determine if any more features exist in thedatabase. Thus, in one aspect, the invention provides a computer programthat identifies open reading frames (ORFs).

A polypeptide or nucleic acid sequence of the invention may be storedand manipulated in a variety of data processor programs in a variety offormats. For example, a sequence can be stored as text in a wordprocessing file, such as MicrosoftWORD or WORDPERFECT or as an ASCIIfile in a variety of database programs familiar to those of skill in theart, such as DB2, SYBASE, or ORACLE. In addition, many computer programsand databases may be used as sequence comparison algorithms,identifiers, or sources of reference nucleotide sequences or polypeptidesequences to be compared to a nucleic acid sequence of the invention.The programs and databases used to practice the invention include, butare not limited to: MacPattern (EMBL), DiscoveryBase (MolecularApplications Group), GeneMine (Molecular Applications Group), Look(Molecular Applications Group), MacLook (Molecular Applications Group),BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol.Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci.USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci.6:237-245, 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE(Molecular Simulations Inc.), Cerius2.DBAccess (Molecular SimulationsInc.), HypoGen (Molecular Simulations Inc.), Insight II, (MolecularSimulations Inc.), Discover (Molecular Simulations Inc.), CHARMm(Molecular Simulations Inc.), Felix (Molecular Simulations Inc.),DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), Modeler (MolecularSimulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer (Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), the MDL Available Chemicals Directory database, the MDL Drug DataReport data base, the Comprehensive Medicinal Chemistry database,Derwent's World Drug Index database, the BioByteMasterFile database, theGenbank database, and the Genseqn database. Many other programs and databases would be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices, and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites, and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated or recombinant nucleic acids thathybridize under stringent conditions to an exemplary sequence of theinvention, e.g., a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, or a nucleic acid that encodes apolypeptide comprising a sequence as set forth in SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, or SEQ ID NO:30, or enzymatically active fragments thereof. Thestringent conditions can be highly stringent conditions, mediumstringent conditions, low stringent conditions, including the high andreduced stringency conditions described herein. In alternativeembodiments, nucleic acids of the invention as defined by their abilityto hybridize under stringent conditions can be between about fiveresidues and the full length of the molecule, e.g., an exemplary nucleicacid of the invention. For example, they can be at least 5, 10, 15, 20,25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300,350, 400 residues in length. Nucleic acids shorter than full length arealso included. These nucleic acids are useful as, e.g., hybridizationprobes, labeling probes, PCR oligonucleotide probes, iRNA (single ordouble stranded), antisense or sequences encoding antibody bindingpeptides (epitopes), motifs, active sites and the like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C. Alternatively, nucleic acids of the invention aredefined by their ability to hybridize under high stringency comprisingconditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS, and arepetitive sequence blocking nucleic acid, such as cot-1 or salmon spermDNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA). In oneaspect, nucleic acids of the invention are defined by their ability tohybridize under reduced stringency conditions comprising 35% formamideat a reduced temperature of 35° C.

Following hybridization, the filter may be washed with 6×SSC, 0.5% SDSat 50° C. These conditions are considered to be “moderate” conditionsabove 25% formamide and “low” conditions below 25% formamide. A specificexample of “moderate” hybridization conditions is when the abovehybridization is conducted at 30% formamide. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 10% formamide.

The temperature range corresponding to a particular level of stringencycan be further narrowed by calculating the purine to pyrimidine ratio ofthe nucleic acid of interest and adjusting the temperature accordingly.Nucleic acids of the invention are also defined by their ability tohybridize under high, medium, and low stringency conditions as set forthin Ausubel and Sambrook. Variations on the above ranges and conditionsare well known in the art. Hybridization conditions are discussedfurther, below.

Oligonucleotides Probes and Methods for Using them

The invention also provides nucleic acid probes for identifying nucleicacids encoding a polypeptide with an aldolase activity. In one aspect,the probe comprises at least 10 consecutive bases of a sequence of theinvention, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, or, a nucleic acid encoding SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30, or enzymatically active fragments thereof. Alternatively, a probeof the invention can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 55, 60,65, 70, 75, 80, 90, 100, 150, about 10 to 50, about 20 to 60 about 30 to70, consecutive bases of a sequence as set forth in a sequence of theinvention. The probes identify a nucleic acid by binding orhybridization. The probes can be used in arrays of the invention, seediscussion below, including, e.g., capillary arrays. The probes of theinvention can also be used to isolate other nucleic acids orpolypeptides.

The probes of the invention can be used to determine whether abiological sample, such as an environmental sample, e.g., a soil sample,contains an organism having a nucleic acid sequence of the invention oran organism from which the nucleic acid was obtained. In suchprocedures, a biological sample potentially harboring the organism fromwhich the nucleic acid was isolated is obtained and nucleic acids areobtained from the sample. The nucleic acids are contacted with the probeunder conditions which permit the probe to specifically hybridize to anycomplementary sequences present in the sample. Where necessary,conditions which permit the probe to specifically hybridize tocomplementary sequences may be determined by placing the probe incontact with complementary sequences from samples known to contain thecomplementary sequence, as well as control sequences which do notcontain the complementary sequence. Hybridization conditions, such asthe salt concentration of the hybridization buffer, the formamideconcentration of the hybridization buffer, or the hybridizationtemperature, may be varied to identify conditions which allow the probeto hybridize specifically to complementary nucleic acids (see discussionon specific hybridization conditions).

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product. Manymethods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel and Sambrook.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). In one aspect, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook (seediscussion on amplification reactions). In such procedures, the nucleicacids in the sample are contacted with the probes, the amplificationreaction is performed, and any resulting amplification product isdetected. The amplification product may be detected by performing gelelectrophoresis on the reaction products and staining the gel with anintercalator such as ethidium bromide. Alternatively, one or more of theprobes may be labeled with a radioactive isotope and the presence of aradioactive amplification product may be detected by autoradiographyafter gel electrophoresis.

Probes derived from sequences near the 3′ or 5′ ends of a nucleic acidsequence of the invention can also be used in chromosome walkingprocedures to identify clones containing additional, e.g., genomicsequences. Such methods allow the isolation of genes which encodeadditional proteins of interest from the host organism.

In one aspect, nucleic acid sequences of the invention are used asprobes to identify and isolate related nucleic acids. In some aspects,the so-identified related nucleic acids may be cDNAs or genomic DNAsfrom organisms other than the one from which the nucleic acid of theinvention was first isolated. In such procedures, a nucleic acid sampleis contacted with the probe under conditions which permit the probe tospecifically hybridize to related sequences. Hybridization of the probeto nucleic acids from the related organism is then detected using any ofthe methods described above.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH2PO4, pH 7.0, 5.0 mMNa2EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×107 cpm (specific activity 4-9×108 cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature (RT) in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash infresh 1×SET at Tm-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, Tm, is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the Tm for a particular probe. The melting temperature of the probemay be calculated using the following exemplary formulas. For probesbetween 14 and 70 nucleotides in length the melting temperature (Tm) iscalculated using the formula: Tm=81.5+16.6(log [Na+])+0.41(fractionG+C)−(600/N) where N is the length of the probe. If the hybridization iscarried out in a solution containing formamide, the melting temperaturemay be calculated using the equation: Tm=81.5+16.6(log[Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N) where N is thelength of the probe. Prehybridization may be carried out in 6×SSC,5×Denhardt's reagent, 0.5% SDS, 100 μg denatured fragmented salmon spermDNA or 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg denaturedfragmented salmon sperm DNA, 50% formamide. Formulas for SSC andDenhardt's and other solutions are listed, e.g., in Sambrook.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the Tm. For shorterprobes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the Tm. In one aspect, hybridizations in6×SSC are conducted at approximately 68° C. In one aspect,hybridizations in 50% formamide containing solutions are conducted atapproximately 42° C. All of the foregoing hybridizations would beconsidered to be under conditions of high stringency.

Following hybridization, the filter is washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examplesof progressively higher stringency condition washes are as follows:2×SSC, 0.1% SDS at room temperature for 15 minutes (low stringency);0.1×SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes.

Nucleic acids which have hybridized to the probe can be identified byautoradiography or other conventional techniques. The above proceduremay be modified to identify nucleic acids having decreasing levels ofhomology to the probe sequence. For example, to obtain nucleic acids ofdecreasing homology to the detectable probe, less stringent conditionsmay be used. For example, the hybridization temperature may be decreasedin increments of 5° C. from 68° C. to 42° C. in a hybridization bufferhaving a Na+ concentration of approximately 1M. Following hybridization,the filter may be washed with 2×SSC, 0.5% SDS at the temperature ofhybridization. These conditions are considered to be “moderate”conditions above 50° C. and “low” conditions below 50° C. An example of“moderate” hybridization conditions is when the above hybridization isconducted at 55° C. An example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

These probes and methods of the invention can be used to isolate nucleicacids having a sequence with at least about 99%, 98%, 97%, at least 95%,at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 65%, at least 60%, at least 55%, or at least 50% homology to anucleic acid sequence of the invention comprising at least about 10, 15,20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, or 500consecutive bases thereof, and the sequences complementary thereto.Homology may be measured using an alignment algorithm, as discussedherein. For example, the homologous polynucleotides may have a codingsequence which is a naturally occurring allelic variant of one of thecoding sequences described herein. Such allelic variants may have asubstitution, deletion or addition of one or more nucleotides whencompared to nucleic acids of the invention.

Additionally, the probes and methods of the invention may be used toisolate nucleic acids which encode polypeptides having at least about99%, at least 95%, at least 90%, at least 85%, at least 80%, at least75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least50% sequence identity (homology) to a polypeptide of the inventioncomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof as determined using a sequence alignmentalgorithm (e.g., such as the FASTA version 3.0t78 algorithm with thedefault parameters, or a BLAST 2.2.2 program with exemplary settings asset forth herein).

Inhibiting Expression of Aldolases and Lyases

The invention provides nucleic acids complementary to (e.g., antisensesequences to) the nucleic acids of the invention, e.g., polynucleotidesencoding proteins of the invention have an aldolase activity, e.g.,aldolase enzyme-encoding nucleic acids. The invention further providesnucleic acids complementary to (e.g., antisense sequences to) aldolasesand lyases.

Antisense sequences are capable of inhibiting the transport, splicing ortranscription of aldolase-encoding genes. The inhibition can be effectedthrough the targeting of genomic DNA or messenger RNA. The transcriptionor function of targeted nucleic acid can be inhibited, for example, byhybridization and/or cleavage. One particularly useful set of inhibitorsprovided by the present invention includes oligonucleotides which areable to either bind aldolase gene or message, in either case preventingor inhibiting the production or function of aldolase enzyme. Theassociation can be though sequence specific hybridization. Anotheruseful class of inhibitors includes oligonucleotides which causeinactivation or cleavage of aldolase message. The oligonucleotide canhave enzyme activity which causes such cleavage, such as ribozymes. Theoligonucleotide can be chemically modified or conjugated to an enzyme orcomposition capable of cleaving the complementary nucleic acid. One mayscreen a pool of many different such oligonucleotides for those with thedesired activity.

The compositions of the invention for the inhibition of aldolaseexpression (e.g., antisense, iRNA, ribozymes, antibodies) can be used aspharmaceutical compositions.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingaldolase message which can inhibit aldolase activity by targeting mRNA.Strategies for designing antisense oligonucleotides are well describedin the scientific and patent literature, and the skilled artisan candesign such aldolase oligonucleotides using the novel reagents of theinvention. For example, gene walking/RNA mapping protocols to screen foreffective antisense oligonucleotides are well known in the art, see,e.g., Ho (2000) Methods Enzymol. 314:168-183, describing an RNA mappingassay, which is based on standard molecular techniques to provide aneasy and reliable method for potent antisense sequence selection. Seealso Smith (2000) Eur. J. Pharm. Sci. 11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl)glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisensealdolase sequences of the invention (see, e.g., Gold (1995) J. of Biol.Chem. 270:13581-13584).

Inhibitory Ribozymes

The invention provides for with ribozymes capable of binding aldolasemessage which can inhibit aldolase enzyme activity by targeting mRNA.Strategies for designing ribozymes and selecting the aldolase-specificantisense sequence for targeting are well described in the scientificand patent literature, and the skilled artisan can design such ribozymesusing the novel reagents of the invention. Ribozymes act by binding to atarget RNA through the target RNA binding portion of a ribozyme which isheld in close proximity to an enzymatic portion of the RNA that cleavesthe target RNA. Thus, the ribozyme recognizes and binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cleave and inactivate the target RNA. Cleavage ofa target RNA in such a manner will destroy its ability to directsynthesis of an encoded protein if the cleavage occurs in the codingsequence. After a ribozyme has bound and cleaved its RNA target, it istypically released from that RNA and so can bind and cleave new targetsrepeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif,but may also be formed in the motif of a hairpin, hepatitis delta virus,group I intron or RNaseP-like RNA (in association with an RNA guidesequence). Examples of such hammerhead motifs are described by Rossi(1992) Aids Research and Human Retroviruses 8:183; hairpin motifs byHampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.18:299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry31:16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849; and thegroup I intron by Cech U.S. Pat. No. 4,987,071. The recitation of thesespecific motifs is not intended to be limiting; those skilled in the artwill recognize that an enzymatic RNA molecule of this invention has aspecific substrate binding site complementary to one or more of thetarget gene RNA regions, and has nucleotide sequence within orsurrounding that substrate binding site which imparts an RNA cleavingactivity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising an aldolase sequence of theinvention. The RNAi molecule comprises a double-stranded RNA (dsRNA)molecule. The RNAi can inhibit expression of an aldolase gene. In oneaspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length. While the invention is not limited byany particular mechanism of action, the RNAi can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed todouble-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's of theinvention. The process may be practiced in vitro, ex vivo or in vivo. Inone aspect, the RNAi molecules of the invention can be used to generatea loss-of-function mutation in a cell, an organ or an animal. Methodsfor making and using RNAi molecules for selectively degrade RNA are wellknown in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824;6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding an aldolase enzyme. Thesemethods can be repeated or used in various combinations to generatealdolase enzymes having an altered or different activity or an alteredor different stability from that of an aldolase encoded by the templatenucleic acid. These methods also can be repeated or used in variouscombinations, e.g., to generate variations in gene/message expression,message translation or message stability. In another aspect, the geneticcomposition of a cell is altered by, e.g., modification of a homologousgene ex vivo, followed by its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods.

Methods for random mutation of genes are well known in the art, see,e.g., U.S. Pat. No. 5,830,696. For example, mutagens can be used torandomly mutate a gene. Mutagens include, e.g., ultraviolet light orgamma irradiation, or a chemical mutagen, e.g., mitomycin, nitrous acid,photoactivated psoralens, alone or in combination, to induce DNA breaksamenable to repair by recombination. Other chemical mutagens include,for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine orformic acid. Other mutagens are analogues of nucleotide precursors,e.g., nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Crameri et al. (1996) “Improved green fluorescent protein bymolecular evolution using DNA shuffling” Nature Biotechnology14:315-319; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) “Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols used in the methods of the invention include pointmismatch repair (Kramer (1984) “Point Mismatch Repair” Cell 38:879-887),mutagenesis using repair-deficient host strains (Carter et al. (1985)“Improved oligonucleotide site-directed mutagenesis using M13 vectors”Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

See also U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Certain U.S. applications provide additional details regarding variousdiversity generating methods, including “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999, (U.S. Ser. No. 09/407,800);“EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCERECOMBINATION” by del Cardayre et al., filed Jul. 15, 1998 (U.S. Ser.No. 09/166,188), and Jul. 15, 1999 (U.S. Ser. No. 09/354,922);“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,filed Sep. 28, 1999 (U.S. Ser. No. 09/408,392), and “OLIGONUCLEOTIDEMEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al., filed Jan. 18,2000 (PCT/US00/01203); “USE OF CODON-VARIED OLIGONUCLEOTIDE SYNTHESISFOR SYNTHETIC SHUFFLING” by Welch et al., filed Sep. 28, 1999 (U.S. Ser.No. 09/408,393); “METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES& POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” by Selifonov et al.,filed Jan. 18, 2000, (PCT/US00/01202) and, e.g. “METHODS FOR MAKINGCHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIREDCHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000 (U.S. Ser. No.09/618,579); “METHODS OF POPULATING DATA STRUCTURES FOR USE INEVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan. 18, 2000(PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATEDRECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” by Affholter, filedSep. 6, 2000 (U.S. Ser. No. 09/656,549).

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or acombination thereof are used to modify the nucleic acids of theinvention to generate aldolases with new or altered properties (e.g.,activity under highly acidic or alkaline conditions, high temperatures,and the like). Polypeptides encoded by the modified nucleic acids can bescreened for an activity before testing for an aldolase or otheractivity. Any testing modality or protocol can be used, e.g., using acapillary array platform. See, e.g., U.S. Pat. Nos. 6,280,926;5,939,250.

Saturation Mutagenesis, or, GSSM

In one aspect of the invention, non-stochastic gene modification, a“directed evolution process,” is used to generate aldolases with new oraltered properties. Variations of this method have been termed “genesite-saturation mutagenesis,” “site-saturation mutagenesis,” “saturationmutagenesis” or simply “GSSM.” It can be used in combination with othermutagenization processes. See, e.g., U.S. Pat. Nos. 6,171,820;6,238,884. In one aspect, GSSM comprises providing a templatepolynucleotide and a plurality of oligonucleotides, wherein eacholigonucleotide comprises a sequence homologous to the templatepolynucleotide, thereby targeting a specific sequence of the templatepolynucleotide, and a sequence that is a variant of the homologous gene;generating progeny polynucleotides comprising non-stochastic sequencevariations by replicating the template polynucleotide with theoligonucleotides, thereby generating polynucleotides comprisinghomologous gene sequence variations.

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, so as togenerate a set of progeny polypeptides in which a full range of singleamino acid substitutions is represented at each amino acid position,e.g., an amino acid residue in an enzyme active site or ligand bindingsite targeted to be modified. These oligonucleotides can comprise acontiguous first homologous sequence, a degenerate N,N,G/T sequence,and, optionally, a second homologous sequence. The downstream progenytranslational products from the use of such oligonucleotides include allpossible amino acid changes at each amino acid site along thepolypeptide, because the degeneracy of the N,N,G/T sequence includescodons for all 20 amino acids. In one aspect, one such degenerateoligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) isused for subjecting each original codon in a parental polynucleotidetemplate to a full range of codon substitutions. In another aspect, atleast two degenerate cassettes are used—either in the sameoligonucleotide or not, for subjecting at least two original codons in aparental polynucleotide template to a full range of codon substitutions.For example, more than one N,N,G/T sequence can be contained in oneoligonucleotide to introduce amino acid mutations at more than one site.This plurality of N,N,G/T sequences can be directly contiguous, orseparated by one or more additional nucleotide sequence(s). In anotheraspect, oligonucleotides serviceable for introducing additions anddeletions can be used either alone or in combination with the codonscontaining an N,N,G/T sequence, to introduce any combination orpermutation of amino acid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position X 100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,aldolase) molecules such that all 20 natural amino acids are representedat the one specific amino acid position corresponding to the codonposition mutagenized in the parental polynucleotide (other aspects useless than all 20 natural combinations). The 32-fold degenerate progenypolypeptides generated from each saturation mutagenesis reaction vesselcan be subjected to clonal amplification (e.g. cloned into a suitablehost, e.g., E. coli host, using, e.g., an expression vector) andsubjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedaldolase activity under alkaline or acidic conditions), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined—6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In another aspect, site-saturation mutagenesis can be used together withanother stochastic or non-stochastic means to vary sequence, e.g.,synthetic ligation reassembly (see below), shuffling, chimerization,recombination and other mutagenizing processes and mutagenizing agents.This invention provides for the use of any mutagenizing process(es),including saturation mutagenesis, in an iterative manner.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate aldolases with new or altered properties. SLR is amethod of ligating oligonucleotide fragments togethernon-stochastically. This method differs from stochastic oligonucleotideshuffling in that the nucleic acid building blocks are not shuffled,concatenated or chimerized randomly, but rather are assemblednon-stochastically. See, e.g., U.S. patent application Ser. No.09/332,835 entitled “Synthetic Ligation Reassembly in DirectedEvolution” and filed on Jun. 14, 1999 (“U.S. Ser. No. 09/332,835”). Inone aspect, SLR comprises the following steps: (a) providing a templatepolynucleotide, wherein the template polynucleotide comprises sequenceencoding a homologous gene; (b) providing a plurality of building blockpolynucleotides, wherein the building block polynucleotides are designedto cross-over reassemble with the template polynucleotide at apredetermined sequence, and a building block polynucleotide comprises asequence that is a variant of the homologous gene and a sequencehomologous to the template polynucleotide flanking the variant sequence;(c) combining a building block polynucleotide with a templatepolynucleotide such that the building block polynucleotide cross-overreassembles with the template polynucleotide to generate polynucleotidescomprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10¹⁰⁰ different chimeras. SLR can be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled.

In one aspect of this method, the sequences of a plurality of parentalnucleic acid templates are aligned in order to select one or moredemarcation points. The demarcation points can be located at an area ofhomology, and are comprised of one or more nucleotides. Thesedemarcation points are preferably shared by at least two of theprogenitor templates. The demarcation points can thereby be used todelineate the boundaries of oligonucleotide building blocks to begenerated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in anotherembodiment, the assembly order (i.e. the order of assembly of eachbuilding block in the 5′ to 3 sequence of each finalized chimericnucleic acid) in each combination is by design (or non-stochastic) asdescribed above. Because of the non-stochastic nature of this invention,the possibility of unwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecularly homologousdemarcation points and thus to allow an increased number of couplings tobe achieved among the building blocks, which in turn allows a greaternumber of progeny chimeric molecules to be generated.

In another aspect, the synthetic nature of the step in which thebuilding blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g. by mutagenesis) or in an in vivoprocess (e.g. by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

In one aspect, a nucleic acid building block is used to introduce anintron. Thus, functional introns are introduced into a man-made genemanufactured according to the methods described herein. The artificiallyintroduced intron(s) can be functional in a host cells for gene splicingmuch in the way that naturally-occurring introns serve functionally ingene splicing.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate aldolases with new oraltered properties. Optimized directed evolution is directed to the useof repeated cycles of reductive reassortment, recombination andselection that allow for the directed molecular evolution of nucleicacids through recombination. Optimized directed evolution allowsgeneration of a large population of evolved chimeric sequences, whereinthe generated population is significantly enriched for sequences thathave a predetermined number of crossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found in U.S. Ser. No.09/332,835. The number of oligonucleotides generated for each parentalvariant bears a relationship to the total number of resulting crossoversin the chimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding an polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide preferablyincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that aoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. One can calculate such a probability density function,and thus enrich the chimeric progeny population for a predeterminednumber of crossover events resulting from a particular ligationreaction. Moreover, a target number of crossover events can bepredetermined, and the system then programmed to calculate the startingquantities of each parental oligonucleotide during each step in theligation reaction to result in a probability density function thatcenters on the predetermined number of crossover events.

Determining Crossover Events

Embodiments of the invention include a system and software that receivea desired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLAB® (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example a nucleic acid (or, the nucleic acid) responsiblefor an altered aldolase phenotype is identified, re-isolated, againmodified, re-tested for activity. This process can be iterativelyrepeated until a desired phenotype is engineered. For example, an entirebiochemical anabolic or catabolic pathway can be engineered into a cell,including aldolase activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new aldolase phenotype), itcan be removed as a variable by synthesizing larger parentaloligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,aldolase enzymes, and the like. In vivo shuffling can be performedutilizing the natural property of cells to recombine multimers. Whilerecombination in vivo has provided the major natural route to moleculardiversity, genetic recombination remains a relatively complex processthat involves 1) the recognition of homologies; 2) strand cleavage,strand invasion, and metabolic steps leading to the production ofrecombinant chiasma; and finally 3) the resolution of chiasma intodiscrete recombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In one aspect, the invention provides a method for producing a hybridpolynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology into a suitable host cell. The regions of partialsequence homology promote processes which result in sequencereorganization producing a hybrid polynucleotide. The term “hybridpolynucleotide”, as used herein, is any nucleotide sequence whichresults from the method of the present invention and contains sequencefrom at least two original polynucleotide sequences. Such hybridpolynucleotides can result from intermolecular recombination eventswhich promote sequence integration between DNA molecules. In addition,such hybrid polynucleotides can result from intramolecular reductivereassortment processes which utilize repeated sequences to alter anucleotide sequence within a DNA molecule.

Producing Sequence Variants

The invention also provides methods of making sequence variants of thenucleic acid and polypeptide (e.g., aldolase) sequences of the inventionor isolating aldolase sequence variants using the nucleic acids andpolypeptides of the invention. In one aspect, the invention provides forvariants of an aldolase gene of the invention, which can be altered byany means, including, e.g., random or stochastic methods, or,non-stochastic, or “directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl2, MnCl2, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl2, 0.5 mMMnCl2, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids isevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/μl in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In someembodiments, random mutations in a sequence of interest are generated bypropagating the sequence of interest in a bacterial strain, such as anE. coli strain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described, e.g., inPCT Publication No. WO 91/16427.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815.

In some embodiments, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described,e.g., in Delegrave (1993) Biotechnology Res. 11: 1548-1552. Random andsite-directed mutagenesis are described, e.g., in Arnold (1993) CurrentOpinion in Biotechnology 4:450-455.

In some embodiments, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in, e.g., U.S.Pat. Nos. 5,965,408; 5,939,250.

The invention also provides variants of polypeptides of the inventioncomprising sequences in which one or more of the amino acid residues(e.g., of an exemplary polypeptide of the invention) are substitutedwith a conserved or non-conserved amino acid residue (e.g., a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code. Conservative substitutions arethose that substitute a given amino acid in a polypeptide by anotheramino acid of like characteristics. Thus, polypeptides of the inventioninclude those with conservative substitutions of sequences of theinvention, including but not limited to the following replacements:replacements of an aliphatic amino acid such as Alanine, Valine, Leucineand Isoleucine with another aliphatic amino acid; replacement of aSerine with a Threonine or vice versa; replacement of an acidic residuesuch as Aspartic acid and Glutamic acid with another acidic residue;replacement of a residue bearing an amide group, such as Asparagine andGlutamine, with another residue bearing an amide group; exchange of abasic residue such as Lysine and Arginine with another basic residue;and replacement of an aromatic residue such as Phenylalanine, Tyrosinewith another aromatic residue. Other variants are those in which one ormore of the amino acid residues of the polypeptides of the inventionincludes a substituent group.

Other variants within the scope of the invention are those in which thepolypeptide is associated with another compound, such as a compound toincrease the half-life of the polypeptide, for example, polyethyleneglycol.

Additional variants within the scope of the invention are those in whichadditional amino acids are fused to the polypeptide, such as a leadersequence, a secretory sequence, a proprotein sequence or a sequencewhich facilitates purification, enrichment, or stabilization of thepolypeptide.

In some aspects, the variants, fragments, derivatives and analogs of thepolypeptides of the invention retain the same biological function oractivity as the exemplary polypeptides, e.g., an aldolase activity, asdescribed herein. In other aspects, the variant, fragment, derivative,or analog includes a proprotein, such that the variant, fragment,derivative, or analog can be activated by cleavage of the proproteinportion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying aldolase-encoding nucleicacids to modify codon usage. In one aspect, the invention providesmethods for modifying codons in a nucleic acid encoding an aldolase toincrease or decrease its expression in a host cell. The invention alsoprovides nucleic acids encoding an aldolase modified to increase itsexpression in a host cell, aldolase enzymes so modified, and methods ofmaking the modified aldolase enzymes. The method comprises identifying a“non-preferred” or a “less preferred” codon in aldolase-encoding nucleicacid and replacing one or more of these non-preferred or less preferredcodons with a “preferred codon” encoding the same amino acid as thereplaced codon and at least one non-preferred or less preferred codon inthe nucleic acid has been replaced by a preferred codon encoding thesame amino acid. A preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli and Pseudomonas fluorescens; gram positive bacteria, such asStreptomyces diversa, Lactobacillus gasseri, Lactococcus lactis,Lactococcus cremoris, Bacillus subtilis. Exemplary host cells alsoinclude eukaryotic organisms, e.g., various yeast, such as Saccharomycessp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha,Aspergillus niger, and mammalian cells and cell lines and insect cellsand cell lines. Thus, the invention also includes nucleic acids andpolypeptides optimized for expression in these organisms and species.

For example, the codons of a nucleic acid encoding an aldolase isolatedfrom a bacterial cell are modified such that the nucleic acid isoptimally expressed in a bacterial cell different from the bacteria fromwhich the aldolase was derived, a yeast, a fungi, a plant cell, aninsect cell or a mammalian cell. Methods for optimizing codons are wellknown in the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int.J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect.Immun. 69:7250-7253, describing optimizing codons in mouse systems;Outchkourov (2002) Protein Expr. Purif. 24:18-24, describing optimizingcodons in yeast; Feng (2000) Biochemistry 39:15399-15409, describingoptimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif.20:252-264, describing optimizing codon usage that affects secretion inE. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide, an expression cassette or vector or a transfectedor transformed cell of the invention. The transgenic non-human animalscan be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice,comprising the nucleic acids of the invention. These animals can beused, e.g., as in vivo models to study aldolase activity, or, as modelsto screen for modulators of aldolase activity in vivo. The codingsequences for the polypeptides to be expressed in the transgenicnon-human animals can be designed to be constitutive, or, under thecontrol of tissue-specific, developmental-specific or inducibletranscriptional regulatory factors. Transgenic non-human animals can bedesigned and generated using any method known in the art; see, e.g.,U.S. Pat. Nos. 6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166;6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698;5,639,940; 5,573,933; 5,387,742; 5,087,571, describing making and usingtransformed cells and eggs and transgenic mice, rats, rabbits, sheep,pigs and cows. See also, e.g., Pollock (1999) J. Immunol. Methods231:147-157, describing the production of recombinant proteins in themilk of transgenic dairy animals; Baguisi (1999) Nat. Biotechnol.17:456-461, demonstrating the production of transgenic goats. U.S. Pat.No. 6,211,428, describes making and using transgenic non-human mammalswhich express in their brains a nucleic acid construct comprising a DNAsequence. U.S. Pat. No. 5,387,742, describes injecting clonedrecombinant or synthetic DNA sequences into fertilized mouse eggs,implanting the injected eggs in pseudo-pregnant females, and growing toterm transgenic mice whose cells express proteins related to thepathology of Alzheimer's disease. U.S. Pat. No. 6,187,992, describesmaking and using a transgenic mouse whose genome comprises a disruptionof the gene encoding amyloid precursor protein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express or to be unable to express analdolase.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., an aldolase), an expression cassette orvector or a transfected or transformed cell of the invention. Theinvention also provides plant products, e.g., oils, seeds, leaves,extracts and the like, comprising a nucleic acid and/or a polypeptide(e.g., an aldolase) of the invention. The transgenic plant can bedicotyledonous (a dicot) or monocotyledonous (a monocot). The inventionalso provides methods of making and using these transgenic plants andseeds. The transgenic plant or plant cell expressing a polypeptide ofthe invention may be constructed in accordance with any method known inthe art. See, for example, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's aldolase production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on oil-seed containing plants, such assoybeans, rapeseed, sunflower seeds, sesame and peanuts. Nucleic acidsof the invention can be used to manipulate metabolic pathways of a plantin order to optimize or alter host's expression of aldolase. The canchange aldolase activity in a plant. Alternatively, an aldolase of theinvention can be used in production of a transgenic plant to produce acompound not naturally produced by that plant. This can lower productioncosts or create a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. For example, aconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation and microinjection ofplant cell protoplasts, or the constructs can be introduced directly toplant tissue using ballistic methods, such as DNA particle bombardment.For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730,describing particle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with nucleicacids, e.g., an expression construct. Although plant regeneration fromprotoplasts is not easy with cereals, plant regeneration is possible inlegumes using somatic embryogenesis from protoplast derived callus.Organized tissues can be transformed with naked DNA using gene guntechnique, where DNA is coated on tungsten microprojectiles, shot1/100th the size of cells, which carry the DNA deep into cells andorganelles. Transformed tissue is then induced to regenerate, usually bysomatic embryogenesis. This technique has been successful in severalcereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci. USA80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol.32:1135-1148, discussing T-DNA integration into genomic DNA. See alsoD'Halluin, U.S. Pat. No. 5,712,135, describing a process for the stableintegration of a DNA comprising a gene that is functional in a cell of acereal, or other monocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering behavior is altered) canbe enhanced when both parental plants express the polypeptides (e.g., analdolase) of the invention. The desired effects can be passed to futureplant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants (e.g., as transgenic plants), such as oil-seedcontaining plants, e.g., soybeans, rapeseed, sunflower seeds, sesame andpeanuts. The nucleic acids of the invention can be expressed in plantswhich contain fiber cells, including, e.g., cotton, silk cotton tree(Kapok, Ceiba pentandra), desert willow, creosote bush, winterfat,balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca and flax. Inalternative embodiments, the transgenic plants of the invention can bemembers of the genus Gossypium, including members of any Gossypiumspecies, such as G. arboreum; G. herbaceum, G. barbadense, and G.hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides (e.g., an aldolase orantibody) of the invention. For example, see Palmgren (1997) TrendsGenet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing humanmilk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas1′,2′) promoterwith Agrobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

Polypeptides and Peptides

The invention provides isolated or recombinant polypeptides having asequence identity (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more, or complete (100%) sequence identity) to an exemplarysequence of the invention, e.g., SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30,or enzymatically active fragments thereof. As discussed above, theidentity can be over the full length of the polypeptide, or, theidentity can be over a subsequence thereof, e.g., a region of at leastabout 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700 or more residues. Polypeptides of the invention canalso be shorter than the full length of exemplary polypeptides (e.g.,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, or SEQ ID NO:30, or enzymatically active fragmentsthereof, etc.). In alternative embodiment, the invention providespolypeptides (peptides, fragments) ranging in size between about 5 andthe full length of a polypeptide, e.g., a polypeptide of the inventionhaving an aldolase activity, such as a aldolase enzyme; exemplary sizesbeing of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400 or moreresidues, e.g., contiguous residues of the exemplary aldolases of theinvention, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, or enzymaticallyactive fragments thereof, etc. Peptides of the invention can be usefulas, e.g., labeling probes, antigens, toleragens, motifs, aldolase activesites.

In one aspect, the polypeptide has an aldolase activity. As used herein,aldolase activity includes any aldolase or lyase activity. The enzymesof the invention can have the activity of any aldolase or lyase. Forexample, the aldolases of the invention can catalyze C—C bond formation,and, in one aspect, in a highly stereoselective way. As another example,a polypeptide of the invention can have a 2-deoxyribose-5-phosphatealdolase (DERA) activity, which in one aspect comprises catalysis of thereversible aldol reaction between acetaldehyde andD-glyceraldehyde-3-phosphate to generate D-2-deoxyribose-5-phosphate.DERA aldolase activity of the invention can catalyze the reversibleasymmetric aldol addition reaction of two aldehydes. In one aspect, analdolase of the invention can accept a 3-azidopropinaldehyde as asubstrate in a sequential asymmetric aldol reaction to form adeoxy-azidoethyl pyranose, which is a precursor to the correspondinglactone and atorvastatin (LIPITOR™). In another aspect,2-methyl-substituted aldehydes act as substrates for aldolases of theinvention (see, e.g., DeSantis (2003) Bioorg. Med. Chem. 11:43-52). Inone aspect, an aldolase of the invention can have aD-2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase activity, e.g., tocatalyze a reversible aldol reaction using a D-configurated KDPG assubstrate. In another aspect, an aldolase of the invention is capable ofaccepting both D- and L-glyceraldehyde in the non-phosphorylated form assubstrates for a reversible aldol reaction (see, e.g., Fong (2000) Chem.Biol. 7:873-83). In one aspect, an aldolase of the invention can have acatalytic activity toward enantiomeric substrates such asN-acetyl-L-mannosamine and L-arabinose to produce, e.g., an L-sialicacid or an L-KDO, the mirror-image sugars of the corresponding naturallyoccurring D-sugars (see, e.g., Wada (2003) Bioorg. Med. Chem.11:2091-2098). In one aspect, an aldolase of the invention can have a4-hydroxy-2-oxoglutarate aldolase activity, a fructose-1,6-bisphosphatealdolase (FBP-aldolase) activity, a tagatose-1,6-bisphosphate (TBP)aldolase activity or a 1-rhamnulose-1-phosphate aldolase activity (see,e.g., Schoevaart (2000) Biotechnol. Bioeng. 70:349-352). In one aspect,a FBP-aldolase activity of the invention catalyses the reversiblecondensation of dihydroxyacetone phosphate (DHAP) and glyceraldehydephosphate (G3P) to form fructose bisphosphate (FBP). In one aspect, analdolase of the invention can have broad substrate specificity withrespect to its reverse reaction, e.g., the condensation of an aldosewith pyruvate to form a wide range of 2-keto-3-deoxy-onic acids,including 2-keto-3-deoxy-nonulosonic acid, 2-keto-3-deoxy-octulosonicacid, 2-keto-3-deoxy-heptulosonic acid, and/or2-keto-3-deoxy-hexylosonic acid. In one aspect, aldolases of theinvention can process 2-keto-3-deoxy-onic acids to high-carbon 2-deoxyaldoses. In one aspect, aldolases of the invention can catalyzestereo-selective synthesis of sugars and compositions of mattercomprising, e.g., arabinohexylose, xyloheptulose, threohexylose, andxylohexylose. The methods and polypeptides of the invention can be usedto produce substantially optically pure sugars using racemic substrates.C-alkyl and N-containing sugars can also be produced using methods andpolypeptides of the invention. The methods and polypeptides of theinvention can be used to make disubstituted dihydroxypyrrolidines ordisubstituted-azafuranoses (azasugars derived from pyrrolidines), e.g.,2-methyl-5-hydroxymethyl- and 2,5-dimethyl-3,4-dihydroxypyrrolidines,and a 5-azido-5-deoxyhexylose-1-phosphate, e.g., in a protocolcomprising mixing a 2-azido-substituted-propionaldehyde anddihydroxyacetone phosphate in the presence of a catalytic amount of analdolase of the invention. In one aspect of the invention, mixtures ofenantiomeric D,L-threo 2-amino-3-hydroxy-3-phenylpropionic acids can bestereoisomerically enriched by contacting the mixture with a polypeptideof the invention having a D-threonine aldolase activity. In one aspect,D- and L-threo 2-amino-3-hydroxy-3-(4-methylsulfonylphenyl) propionicacid are treated with a D-threonine aldolase of the invention to produceL-threo 2-amino-3-hydroxy-3-(4-methylsulfonylphenyl)propionic acid witha high ee.

The processes of the invention can involve relatively mild reactionconditions, high stereoselectivity and/or the minimal use of protectivegroup chemistry. These reactions are reversible, as, in some aspects,can be the activity of an aldolase of the invention. In alternativeaspects, the processes of the invention comprise conditions such that aforward or the reverse reaction is favored, e.g., conditions wheresynthesis becomes favored.

Protocols for screening for aldolase activity (e.g., to determine if apolypeptide has an aldolase activity, e.g., 2-deoxyribose-5-phosphatealdolase (DERA) activity, and is within the scope of the invention) arewell known in the art, see, e.g., U.S. Pat. Nos. 6,441,277; 6,423,834;6,368,839; 5,795,749; 5,585,261; 5,576,426; 5,358,859; 5,352,591;5,346,828; 5,346,828.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3

13) and automated synthesis may be achieved, e.g., using the ABI 431APeptide Synthesizer (Perkin Elmer) in accordance with the instructionsprovided by the manufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has analdolase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH2- for —C(═O)—NH—), aminomethylene (CH2-NH), ethylene, olefin(CH═CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4-), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; K- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

Aldolase Enzymes

The invention provides novel aldolases, nucleic acids encoding them,antibodies that bind them, peptides representing the enzyme's antigenicsites (epitopes) and active sites, and methods for making and usingthem. In one aspect, polypeptides of the invention have an aldolaseactivity, as described above (e.g., catalysis of the formation of acarbon-carbon bond). In alternative aspects, the aldolases of theinvention have activities that have been modified from those of theexemplary aldolases described herein. The invention includes aldolaseswith and without signal sequences and the signal sequences themselves.The invention includes immobilized aldolases, anti-aldolase antibodiesand fragments thereof. The invention includes heterocomplexes, e.g.,fusion proteins, heterodimers, etc., comprising the aldolases of theinvention.

Determining peptides representing the enzyme's antigenic sites(epitopes), active sites, binding sites, signal sequences, and the likecan be done by routine screening protocols.

The enzymes of the invention are highly selective catalysts. As withother enzymes, they catalyze reactions with exquisite stereo-, regio-,and chemo-selectivities that are unparalleled in conventional syntheticchemistry. Moreover, the enzymes of the invention are remarkablyversatile. They can be tailored to function in organic solvents, operateat extreme pHs (for example, high pHs and low pHs) extreme temperatures(for example, high temperatures and low temperatures), extreme salinitylevels (for example, high salinity and low salinity), and catalyzereactions with compounds that are structurally unrelated to theirnatural, physiological substrates. Enzymes of the invention can bedesigned to be reactive toward a wide range of natural and unnaturalsubstrates, thus enabling the modification of virtually any organic leadcompound. Enzymes of the invention can also be designed to be highlyenantio- and regio-selective. The high degree of functional groupspecificity exhibited by these enzymes enables one to keep track of eachreaction in a synthetic sequence leading to a new active compound.Enzymes of the invention can also be designed to catalyze many diversereactions unrelated to their native physiological function in nature.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound. The present invention usesselected biocatalysts, i.e., the enzymes of the invention, and reactionconditions that are specific for functional groups that are present inmany starting compounds. Each biocatalyst is specific for one functionalgroup, or several related functional groups, and can react with manystarting compounds containing this functional group. The biocatalyticreactions produce a population of derivatives from a single startingcompound. These derivatives can be subjected to another round ofbiocatalytic reactions to produce a second population of derivativecompounds. Thousands of variations of the original compound can beproduced with each iteration of biocatalytic derivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process that is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active enzyme withina library. The library is characterized by the series of biocatalyticreactions used to produce it, a so-called “biosynthetic history”.Screening the library for biological activities and tracing thebiosynthetic history identifies the specific reaction sequence producingthe active compound. The reaction sequence is repeated and the structureof the synthesized compound determined. This mode of identification,unlike other synthesis and screening approaches, does not requireimmobilization technologies, and compounds can be synthesized and testedfree in solution using virtually any type of screening assay. It isimportant to note, that the high degree of specificity of enzymereactions on functional groups allows for the “tracking” of specificenzymatic reactions that make up the biocatalytically produced library.

The invention also provides methods of discovering new aldolases usingthe nucleic acids, polypeptides and antibodies of the invention. In oneaspect, lambda phage libraries are screened for expression-baseddiscovery of aldolases. Use of lambda phage libraries in screeningallows detection of toxic clones; improved access to substrate; reducedneed for engineering a host, by-passing the potential for any biasresulting from mass excision of the library; and, faster growth at lowclone densities. Screening of lambda phage libraries can be in liquidphase or in solid phase. Screening in liquid phase gives greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility (see discussion of arrays, below). As a result, alibrary of derivative compounds can be produced in a matter of weeks.For further teachings on modification of molecules, including smallmolecules, see PCT/US94/09174.

Aldolase Signal Sequences, Prepro Sequences and Catalytic Domains

The invention provides aldolase signal sequences (e.g., signal peptides(SPs)), prepro sequences and catalytic domains (CDs). The inventionprovides nucleic acids encoding these catalytic domains (CDs), preprosequences and signal sequences (SPs, e.g., a peptide having a sequencecomprising/consisting of amino terminal residues of a polypeptide of theinvention). In one aspect, the invention provides a signal sequencecomprising a peptide comprising/consisting of a sequence as set forth inresidues 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21,1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28,1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37,1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42 or 1 to 43 or more, of apolypeptide of the invention, e.g., SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30, or enzymatically active fragments thereof. For example, anexemplary aldolase signal sequence of the invention is residues 1 to 22of SEQ ID NO:18.

The aldolase signal sequences of the invention can be isolated peptides,or, sequences joined to another aldolase or a non-aldolase polypeptide,e.g., as a fusion protein. In one aspect, the invention providespolypeptides comprising aldolase signal sequences of the invention. Inone aspect, polypeptides comprising aldolase signal sequences of theinvention comprise sequences heterologous to an aldolase of theinvention (e.g., a fusion protein comprising an aldolase signal sequenceof the invention and sequences from another aldolase or a non-aldolaseprotein). In one aspect, the invention provides aldolases of theinvention with heterologous signal sequences, e.g., sequences with ayeast signal sequence. An aldolase of the invention can comprise aheterologous signal sequence, e.g., in a vector, e.g., a pPIC seriesvector (Invitrogen, Carlsbad, Calif.).

In one aspect, the signal sequences of the invention are identifiedfollowing identification of novel aldolase polypeptides. The pathways bywhich proteins are sorted and transported to their proper cellularlocation are often referred to as protein targeting pathways. One of themost important elements in all of these targeting systems is a shortamino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. More than 100 signal sequences for proteins inthis group have been determined. The signal sequences can vary in lengthfrom 13 to 36 amino acid residues. Various methods of recognition ofsignal sequences are known to those of skill in the art. For example, inone aspect, novel aldolase signal peptides are identified by a methodreferred to as SignalP. SignalP uses a combined neural network whichrecognizes both signal peptides and their cleavage sites. (Nielsen, etal., “Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering, vol. 10, no.1, p. 1-6 (1997).

It should be understood that in some aspects aldolases of the inventionmay not have signal sequences. In one aspect, the invention provides thealdolases of the invention lacking all or part of a signal sequence. Inone aspect, the invention provides a nucleic acid sequence encoding asignal sequence from one aldolase operably linked to a nucleic acidsequence of a different aldolase or, optionally, a signal sequence froma non-aldolase protein may be desired.

The invention also provides isolated or recombinant polypeptidescomprising signal sequences (SPs), prepro sequences (PPS) and/orcatalytic domains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toan aldolase) with an SP, PPS and/or CD. The sequence to which the SP,PPS and/or CD are not naturally associated can be on the SP's, PPS'sand/or CD's amino terminal end, carboxy terminal end, and/or on bothends of the SP, PPS and/or CD. In one aspect, the invention provides anisolated or recombinant polypeptide comprising (or consisting of) apolypeptide comprising an SP, PPS and/or CD of the invention with theproviso that it is not associated with any sequence to which it isnaturally associated (e.g., an aldolase sequence). Similarly in oneaspect, the invention provides isolated or recombinant nucleic acidsencoding these polypeptides. Thus, in one aspect, the isolated orrecombinant nucleic acid of the invention comprises coding sequence foran SP, PPS and/or CD of the invention and a heterologous sequence (i.e.,a sequence not naturally associated with the SP, PPS and/or CD of theinvention). The heterologous sequence can be on the 3′ terminal end, 5′terminal end, and/or on both ends of the SP, PPS and/or CD codingsequence.

Hybrid (Chimeric) Aldolases and Peptide Libraries

In one aspect, the invention provides hybrid aldolases and fusionproteins, including peptide libraries, comprising sequences of theinvention. The peptide libraries of the invention can be used to isolatepeptide modulators (e.g., activators or inhibitors) of targets, such asaldolase substrates, receptors, enzymes. The peptide libraries of theinvention can be used to identify formal binding partners of targets,such as ligands, e.g., cytokines, hormones and the like. In one aspect,the invention provides chimeric proteins comprising a signal sequence(SP), a prepro sequence (PPS) and/or catalytic domain (CD) of theinvention and a heterologous sequence (see above).

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

The invention also provides methods for generating “improved” and hybridaldolases using the nucleic acids and polypeptides of the invention. Forexample, the invention provides methods for generating enzymes that haveactivity, e.g., aldolase activity (such as, e.g., catalysis of theformation of a carbon-carbon bond, a 2-deoxyribose-5-phosphate aldolase(DERA) activity) at extreme alkaline pHs and/or acidic pHs, high and lowtemperatures, osmotic conditions and the like. The invention providesmethods for generating hybrid enzymes (e.g., hybrid aldolases).

In one aspect, the methods of the invention produce new hybridpolypeptides by utilizing cellular processes that integrate the sequenceof a first polynucleotide such that resulting hybrid polynucleotidesencode polypeptides demonstrating activities derived from the firstbiologically active polypeptides. For example, the first polynucleotidescan be an exemplary nucleic acid sequence (e.g., SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, etc.) encoding an exemplaryaldolase of the invention (e.g., SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30,or enzymatically active fragments thereof, etc.). The first nucleic acidcan encode an enzyme from one organism that functions effectively undera particular environmental condition, e.g. high salinity. It can be“integrated” with an enzyme encoded by a second polynucleotide from adifferent organism that functions effectively under a differentenvironmental condition, such as extremely high temperatures. Forexample, when the two nucleic acids can produce a hybrid molecule bye.g., recombination and/or reductive reassortment. A hybridpolynucleotide containing sequences from the first and second originalpolynucleotides may encode an enzyme that exhibits characteristics ofboth enzymes encoded by the original polynucleotides. Thus, the enzymeencoded by the hybrid polynucleotide may function effectively underenvironmental conditions shared by each of the enzymes encoded by thefirst and second polynucleotides, e.g., high salinity and extremetemperatures.

Alternatively, a hybrid polypeptide resulting from this method of theinvention may exhibit specialized enzyme activity not displayed in theoriginal enzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding aldolase activities, theresulting hybrid polypeptide encoded by a hybrid polynucleotide can bescreened for specialized activities obtained from each of the originalenzymes. Thus, for example, the aldolase may be screened to ascertainthose chemical functionalities which distinguish the hybrid aldolasefrom the original aldolases, for example, the temperature, pH or saltconcentration at which the hybrid polypeptide functions.

Sources of the polynucleotides to be “integrated” with nucleic acids ofthe invention may be isolated from individual organisms (“isolates”),collections of organisms that have been grown in defined media(“enrichment cultures”), or, uncultivated organisms (“environmentalsamples”). The use of a culture-independent approach to derivepolynucleotides encoding novel bioactivities from environmental samplesis most preferable since it allows one to access untapped resources ofbiodiversity. “Environmental libraries” are generated from environmentalsamples and represent the collective genomes of naturally occurringorganisms archived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample that may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest (e.g., a2-deoxyribose-5-phosphate aldolase (DERA) activity). Potential pathwaysencoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions that promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

The microorganisms from which hybrid polynucleotides may be preparedinclude prokaryotic microorganisms, such as Eubacteria andArchaebacteria, and lower eukaryotic microorganisms such as fungi, somealgae and protozoa. Polynucleotides may be isolated from environmentalsamples. Nucleic acid may be recovered without culturing of an organismor recovered from one or more cultured organisms. In one aspect, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles. Inone aspect, polynucleotides encoding aldolase enzymes isolated fromextremophilic microorganisms are used to make hybrid enzymes. Suchenzymes may function at temperatures above 100° C. in, e.g., terrestrialhot springs and deep sea thermal vents, at temperatures below 0° C. in,e.g., arctic waters, in the saturated salt environment of, e.g., theDead Sea, at pH values around 0 in, e.g., coal deposits and geothermalsulfur-rich springs, or at pH values greater than 11 in, e.g., sewagesludge. For example, aldolases cloned and expressed from extremophilicorganisms can show high activity throughout a wide range of temperaturesand pHs.

Polynucleotides selected and isolated as described herein, including atleast one nucleic acid of the invention, are introduced into a suitablehost cell. A suitable host cell is any cell that is capable of promotingrecombination and/or reductive reassortment. The selectedpolynucleotides can be in a vector that includes appropriate controlsequences. The host cell can be a higher eukaryotic cell, such as amammalian cell, or a lower eukaryotic cell, such as a yeast cell, orpreferably, the host cell can be a prokaryotic cell, such as a bacterialcell. Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-Dextran mediated transfection,or electroporation.

Exemplary hosts include, e.g., bacterial cells, such as E. coli,Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells suchas CHO, COS or Bowes melanoma; adenoviruses; and plant cells. Theselection of an appropriate host for recombination and/or reductivereassortment or just for expression of recombinant protein is deemed tobe within the scope of those skilled in the art from the teachingsherein. Mammalian cell culture systems that can be employed forrecombination and/or reductive reassortment or just for expression ofrecombinant protein include, e.g., the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants”, the C127, 3T3, CHO, HeLa and BHKcell lines. Mammalian expression vectors can comprise an origin ofreplication, a suitable promoter and enhancer, and necessary ribosomebinding sites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking non-transcribedsequences. DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

Host cells containing the polynucleotides of interest (for recombinationand/or reductive reassortment or just for expression of recombinantprotein) can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying genes. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan. Theclones which are identified as having the specified enzyme activity maythen be sequenced to identify the polynucleotide sequence encoding anenzyme having the enhanced activity.

In another aspect, the nucleic acids and methods of the presentinvention can be used to generate novel polynucleotides for biochemicalpathways, e.g., pathways from one or more operons or gene clusters orportions thereof. For example, bacteria and many eukaryotes have acoordinated mechanism for regulating genes whose products are involvedin related processes. The genes are clustered, in structures referred toas “gene clusters,” on a single chromosome and are transcribed togetherunder the control of a single regulatory sequence, including a singlepromoter which initiates transcription of the entire cluster. Thus, agene cluster is a group of adjacent genes that are either identical orrelated, usually as to their function.

Gene cluster DNA can be isolated from different organisms and ligatedinto vectors, particularly vectors containing expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affects high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples.“Fosmids,” cosmids or bacterial artificial chromosome (BAC) vectors canbe used as cloning vectors. These are derived from E. coli f-factorwhich is able to stably integrate large segments of genomic DNA. Whenintegrated with DNA from a mixed uncultured environmental sample, thismakes it possible to achieve large genomic fragments in the form of astable “environmental DNA library.” Cosmid vectors were originallydesigned to clone and propagate large segments of genomic DNA. Cloninginto cosmid vectors is described in detail in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress (1989). Once ligated into an appropriate vector, two or morevectors containing different polyketide synthase gene clusters can beintroduced into a suitable host cell. Regions of partial sequencehomology shared by the gene clusters will promote processes which resultin sequence reorganization resulting in a hybrid gene cluster. The novelhybrid gene cluster can then be screened for enhanced activities notfound in the original gene clusters.

Thus, in one aspect, the invention relates to a method for producing abiologically active hybrid polypeptide using a nucleic acid of theinvention and screening the polypeptide for an activity (e.g., enhancedactivity) by:

(1) introducing at least a first polynucleotide (e.g., a nucleic acid ofthe invention) in operable linkage and a second polynucleotide inoperable linkage, said at least first polynucleotide and secondpolynucleotide sharing at least one region of partial sequence homology,into a suitable host cell;

(2) growing the host cell under conditions which promote sequencereorganization resulting in a hybrid polynucleotide in operable linkage;

(3) expressing a hybrid polypeptide encoded by the hybridpolynucleotide;

(4) screening the hybrid polypeptide under conditions which promoteidentification of the desired biological activity (e.g., enhancedaldolase activity); and

(5) isolating the a polynucleotide encoding the hybrid polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

In vivo reassortment can be focused on “inter-molecular” processescollectively referred to as “recombination.” In bacteria it is generallyviewed as a “RecA-dependent” phenomenon. The invention can rely onrecombination processes of a host cell to recombine and re-assortsequences, or the cells' ability to mediate reductive processes todecrease the complexity of quasi-repeated sequences in the cell bydeletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process. Thus, in one aspect of theinvention, using the nucleic acids of the invention novelpolynucleotides are generated by the process of reductive reassortment.The method involves the generation of constructs containing consecutivesequences (original encoding sequences), their insertion into anappropriate vector, and their subsequent introduction into anappropriate host cell. The reassortment of the individual molecularidentities occurs by combinatorial processes between the consecutivesequences in the construct possessing regions of homology, or betweenquasi-repeated units. The reassortment process recombines and/or reducesthe complexity and extent of the repeated sequences, and results in theproduction of novel molecular species.

Various treatments may be applied to enhance the rate of reassortment.These could include treatment with ultra-violet light, or DNA damagingchemicals, and/or the use of host cell lines displaying enhanced levelsof “genetic instability”. Thus the reassortment process may involvehomologous recombination or the natural property of quasi-repeatedsequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. “Quasi-repeats” are repeats that are not restricted totheir original unit structure. Quasi-repeated units can be presented asan array of sequences in a construct; consecutive units of similarsequences. Once ligated, the junctions between the consecutive sequencesbecome essentially invisible and the quasi-repetitive nature of theresulting construct is now continuous at the molecular level. Thedeletion process the cell performs to reduce the complexity of theresulting construct operates between the quasi-repeated sequences. Thequasi-repeated units provide a practically limitless repertoire oftemplates upon which slippage events can occur. The constructscontaining the quasi-repeats thus effectively provide sufficientmolecular elasticity that deletion (and potentially insertion) eventscan occur virtually anywhere within the quasi-repetitive units. When thequasi-repeated sequences are all ligated in the same orientation, forinstance head to tail or vice versa, the cell cannot distinguishindividual units. Consequently, the reductive process can occurthroughout the sequences. In contrast, when for example, the units arepresented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, in one aspect of theinvention, the sequences to be reasserted are in the same orientation.Random orientation of quasi-repeated sequences will result in the lossof reassortment efficiency, while consistent orientation of thesequences will offer the highest efficiency. However, while having fewerof the contiguous sequences in the same orientation decreases theefficiency, it may still provide sufficient elasticity for the effectiverecovery of novel molecules. Constructs can be made with thequasi-repeated sequences in the same orientation to allow higherefficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following: a) Primers that include apoly-A head and poly-T tail which when made single-stranded wouldprovide orientation can be utilized. This is accomplished by having thefirst few bases of the primers made from RNA and hence easily removedRNase H. b) Primers that include unique restriction cleavage sites canbe utilized. Multiple sites, a battery of unique sequences, and repeatedsynthesis and ligation steps would be required. c) The inner few basesof the primer could be thiolated and an exonuclease used to produceproperly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced repetitive index (RI). The re-assortedencoding sequences can then be recovered by amplification. The productsare re-cloned and expressed. The recovery of cloning vectors withreduced RI can be affected by: 1) The use of vectors only stablymaintained when the construct is reduced in complexity. 2) The physicalrecovery of shortened vectors by physical procedures. In this case, thecloning vector would be recovered using standard plasmid isolationprocedures and size fractionated on either an agarose gel, or columnwith a low molecular weight cut off utilizing standard procedures. 3)The recovery of vectors containing interrupted genes which can beselected when insert size decreases. 4) The use of direct selectiontechniques with an expression vector and the appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, this process is not limitedto such nearly identical repeats.

The following is an exemplary method of the invention. Encoding nucleicacid sequences (quasi-repeats) are derived from three (3) species,including a nucleic acid of the invention. Each sequence encodes aprotein with a distinct set of properties, including an enzyme of theinvention. Each of the sequences differs by a single or a few base pairsat a unique position in the sequence. The quasi-repeated sequences areseparately or collectively amplified and ligated into random assembliessuch that all possible permutations and combinations are available inthe population of ligated molecules. The number of quasi-repeat unitscan be controlled by the assembly conditions. The average number ofquasi-repeated units in a construct is defined as the repetitive index(RI). Once formed, the constructs may, or may not be size fractionatedon an agarose gel according to published protocols, inserted into acloning vector, and transfected into an appropriate host cell. The cellsare then propagated and “reductive reassortment” is effected. The rateof the reductive reassortment process may be stimulated by theintroduction of DNA damage if desired. Whether the reduction in RI ismediated by deletion formation between repeated sequences by an“intra-molecular” mechanism, or mediated by recombination-like eventsthrough “inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations. In oneaspect, the method comprises the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact, orcatalyze a particular reaction (e.g., such as catalytic domain of anenzyme) with a predetermined macromolecule, such as for example aproteinaceous receptor, an oligosaccharide, virion, or otherpredetermined compound or structure. The polypeptides, e.g., aldolases,that are identified from such libraries can be used for variouspurposes, e.g., the industrial processes described herein and/or can besubjected to one or more additional cycles of shuffling and/orselection.

In another aspect, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodof the invention can be subjected to agents or processes which promotethe introduction of mutations into the original polynucleotides. Theintroduction of such mutations would increase the diversity of resultinghybrid polynucleotides and polypeptides encoded therefrom. The agents orprocesses which promote mutagenesis can include, but are not limited to:(+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N-3-Adenine (SeeSun and Hurley, (1992); an N-acetylated or deacetylated4′-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See, for example, van de Poll et al. (1992)); or a N-acetylated ordeacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium,a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNAadduct capable of inhibiting DNA replication, such as7-bromomethyl-benz[a]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”), andN-hydroxy-2-amino-1-methyl-6-phenyl]midazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). Especially preferred means for slowing or haltingPCR amplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N-3-Adenine). Particularly encompassed means are DNAadducts or polynucleotides comprising the DNA adducts from thepolynucleotides or polynucleotides pool, which can be released orremoved by a process including heating the solution comprising thepolynucleotides prior to further processing.

Screening Methodologies and “On-Line” Monitoring Devices

In practicing the methods of the invention, and screening for aldolaseactivity in the polypeptides of the invention, a variety of apparatusand methodologies can be used. For example, a variety of apparatus andmethodologies can be used to screen polypeptides for aldolase activity,to screen compounds as potential modulators of activity (e.g.,potentiation or inhibition of aldolase activity), for antibodies thatbind to an aldolase of the invention or have aldolase activity, fornucleic acids that hybridize to a nucleic acid of the invention, and thelike. High throughput screening apparatus can be adapted and used topractice the methods of the invention, see, e.g., U.S. PatentApplication No. 20020001809.

Immobilized Enzyme Solid Supports

The polypeptides of the invention, e.g., antibodies and aldolaseenzymes, fragments thereof and nucleic acids that encode thepolypeptides of the invention (e.g., aldolases) and fragments can beaffixed to a solid support. This is often economical and efficient inthe use of the aldolases in industrial processes. For example, aconsortium or cocktail of aldolase enzymes (or active fragmentsthereof), which are used in a specific chemical reaction, can beattached to a solid support and dunked into a process vat. The enzymaticreaction can occur. Then, the solid support can be taken out of the vat,along with the enzymes affixed thereto, for repeated use. In oneembodiment of the invention, an isolated nucleic acid of the inventionis affixed to a solid support. In another embodiment of the invention,the solid support is selected from the group of a gel, a resin, apolymer, a ceramic, a glass, a microelectrode and any combinationthereof.

For example, solid supports useful in this invention include gels. Someexamples of gels include Sepharose, gelatin, glutaraldehyde,chitosan-treated glutaraldehyde, albumin-glutaraldehyde,chitosan-Xanthan, toyopearl gel (polymer gel), alginate,alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magneticagarose, dextran-agarose, poly(Carbamoyl Sulfonate) hydrogel, BSA-PEGhydrogel, phosphorylated polyvinyl alcohol (PVA),monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.

Another solid support useful in the present invention are resins orpolymers. Some examples of resins or polymers include cellulose,acrylamide, nylon, rayon, polyester, anion-exchange resin, AMBERLITE™XAD-7, AMBERLITE™ XAD-8, AMBERLITE™ IRA-94, AMBERLITE™ IRC-50,polyvinyl, polyacrylic, polymethacrylate, or any combination thereof.

Another type of solid support useful in the present invention isceramic. Some examples include non-porous ceramic, porous ceramic, SiO₂,Al₂O₃. Another type of solid support useful in the present invention isglass. Some examples include non-porous glass, porous glass, aminopropylglass or any combination thereof. Another type of solid support that canbe used is a microelectrode. An example is a polyethyleneimine-coatedmagnetite. Graphitic particles can be used as a solid support. Anotherexample of a solid support is a cell, such as a red blood cell.

Methods of Immobilization

There are many methods that would be known to one of skill in the artfor immobilizing antibodies, enzymes or fragments thereof, or nucleicacids, onto a solid support. Some examples of such methods include,e.g., electrostatic droplet generation, electrochemical means, viaadsorption, via covalent binding, via cross-linking, via a chemicalreaction or process, via encapsulation, via entrapment, via calciumalginate, or via poly (2-hydroxyethyl methacrylate). Like methods aredescribed in Methods in Enzymology, Immobilized Enzymes and Cells, PartC. 1987. Academic Press. Edited by S. P. Colowick and N, O. Kaplan.Volume 136; and Immobilization of Enzymes and Cells. 1997. Humana Press.Ed. G. F. Bickerstaff. Series: Methods in Biotechnology, Ed. J. M.Walker.

Capillary Arrays

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. Capillary arrays, suchas the GIGAMATRIX™, Diversa Corporation, San Diego, Calif.; and arraysdescribed in, e.g., U.S. Patent Application No. 20020080350 A1; WO0231203 A; WO 0244336 A, provide an alternative apparatus for holdingand screening samples. In one aspect, the capillary array includes aplurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The lumen may be cylindrical, square, hexagonal orany other geometric shape so long as the walls form a lumen forretention of a liquid or sample. The capillaries of the capillary arraycan be held together in close proximity to form a planar structure. Thecapillaries can be bound together, by being fused (e.g., where thecapillaries are made of glass), glued, bonded, or clamped side-by-side.Additionally, the capillary array can include interstitial materialdisposed between adjacent capillaries in the array, thereby forming asolid planar device containing a plurality of through-holes.

A capillary array can be formed of any number of individual capillaries,for example, a range from 100 to 4,000,000 capillaries. Further, acapillary array having about 100,000 or more individual capillaries canbe formed into the standard size and shape of a Microtiter® plate forfitment into standard laboratory equipment. The lumens are filledmanually or automatically using either capillary action ormicroinjection using a thin needle. Samples of interest may subsequentlybe removed from individual capillaries for further analysis orcharacterization. For example, a thin, needle-like probe is positionedin fluid communication with a selected capillary to either add orwithdraw material from the lumen.

In a single-pot screening assay, the assay components are mixed yieldinga solution of interest, prior to insertion into the capillary array. Thelumen is filled by capillary action when at least a portion of the arrayis immersed into a solution of interest. Chemical or biologicalreactions and/or activity in each capillary are monitored for detectableevents. A detectable event is often referred to as a “hit”, which canusually be distinguished from “non-hit” producing capillaries by opticaldetection. Thus, capillary arrays allow for massively parallel detectionof “hits”.

In a multi-pot screening assay, a polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component, which is introducedinto at least a portion of a capillary of a capillary array. An airbubble can then be introduced into the capillary behind the firstcomponent. A second component can then be introduced into the capillary,wherein the second component is separated from the first component bythe air bubble. The first and second components can then be mixed byapplying hydrostatic pressure to both sides of the capillary array tocollapse the bubble. The capillary array is then monitored for adetectable event resulting from reaction or non-reaction of the twocomponents.

In a binding screening assay, a sample of interest can be introduced asa first liquid labeled with a detectable particle into a capillary of acapillary array, wherein the lumen of the capillary is coated with abinding material for binding the detectable particle to the lumen. Thefirst liquid may then be removed from the capillary tube, wherein thebound detectable particle is maintained within the capillary, and asecond liquid may be introduced into the capillary tube. The capillaryis then monitored for a detectable event resulting from reaction ornon-reaction of the particle with the second liquid.

Arrays, or “BioChips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof an aldolase gene. One or more, or, all the transcripts of a cell canbe measured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. “Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins.

The present invention can be practiced with any known “array,” alsoreferred to as a “microarray” or “nucleic acid array” or “polypeptidearray” or “antibody array” or “biochip,” or variation thereof. Arraysare generically a plurality of “spots” or “target elements,” each targetelement comprising a defined amount of one or more biological molecules,e.g., oligonucleotides, immobilized onto a defined area of a substratesurface for specific binding to a sample molecule, e.g., mRNAtranscripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated or recombinant antibodies thatspecifically bind to polypeptides of the invention, e.g., an aldolase ofthe invention or other antibodies of the invention (e.g., ananti-idiotype antibody). These antibodies can be used to isolate,identify or quantify the aldolases of the invention or relatedpolypeptides. These antibodies can be used to inhibit the activity of anenzyme of the invention. These antibodies can be used to isolatedpolypeptides related to those of the invention, e.g., related aldolaseenzymes.

The antibodies can be used in immunoprecipitation, staining (e.g.,FACS), immunoaffinity columns, and the like. If desired, nucleic acidsequences encoding for specific antigens can be generated byimmunization followed by isolation of polypeptide or nucleic acid,amplification or cloning and immobilization of polypeptide onto an arrayof the invention.

Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

The antibodies of the invention can be used to detect or measure theamount of an aldolase in a sample, e.g., a serum aldolase. Normal serumaldolase levels are <6 U/L. High levels of aldolase are found inprogressive Duchenne muscular dystrophy (MD), in carriers of MD, inlimb-girdle dystrophy and other dystrophies, in dermatomyositis,polymyositis, and trichinosis. In the progressive dystrophies, aldolaselevels may be 10-15 times normal when muscle mass is relatively intact,as in early stages of the disease. When advanced muscle wasting ispresent, values decline. Thus, the antibodies of the invention can beused in the treatment, diagnosis or prognosis of conditions or diseases.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides can be used to generate antibodies which bindspecifically to the polypeptides of the invention. The resultingantibodies may be used in immunoaffinity chromatography procedures toisolate or purify the polypeptide or to determine whether thepolypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the invention.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention.After a wash to remove non-specifically bound proteins, the specificallybound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to an animal, forexample, a nonhuman. The antibody so obtained will then bind thepolypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptide can be used to generate antibodies which maybind to the whole native polypeptide. Such antibodies can then be usedto isolate the polypeptide from cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the invention. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention may beused in screening for similar polypeptides from other organisms andsamples. In such techniques, polypeptides from the organism arecontacted with the antibody and those polypeptides which specificallybind the antibody are detected. Any of the procedures described abovemay be used to detect antibody binding.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, polypeptides (e.g.,aldolases) and/or antibodies of the invention. The kits also can containinstructional material teaching the methodologies and industrial uses ofthe invention, as described herein.

Preparation of β,δ-Dihydroxyheptanoic Acid Side Chains

The invention provides compositions and methods for using a2-deoxyribose-5-phosphate aldolase (DERA) in a process to prepare achiral β,δ-dihydroxyheptanoic acid side chain. Any DERA or equivalentaldolase, or enzyme or other polypeptide having a similar activity,natural or synthetic (e.g., catalytic antibodies, see, e.g., U.S. Pat.Nos. 6,368,839; 5,733,757), including an enzyme of the invention, can beused. The chemoenzymatic methods of the invention can use anypolypeptide having an aldolase activity (e.g., an enzyme, a catalyticantibody), e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, including a polypeptide of the invention having an aldolaseactivity, e.g., the exemplary SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,SEQ ID NO:22. Also see, e.g., U.S. Pat. No. 5,795,749, describing how tomake, isolate and test for DERA activity; U.S. Pat. Nos. 6,423,834;6,441,277.

For example, DERA activity can be assayed with a coupled enzymaticsystem where 0.5 mM of 2-deoxyribose-5-phosphate, 0.12 mM NADH, and amixture of glycerophosphate dehydrogenase and triose phosphate isomeraseare incubated in triethanolamine buffer (50 mM, pH 7.5) at 25° C. Theassay can be initiated by addition of DERA, and the decrease in theabsorbance at 340 nm is monitored. The extinction coefficient for NADHis taken as 6.22.times.10³M⁻¹cm⁻¹. Protein concentration can be measuredby a Bradford assay (e.g., a Coomassie Plus Kit Reagent from PierceCo.).

The invention provides novel routes for the chemoenzymatic production ofchiral β,δ-dihydroxyheptanoic acid side chains, including(R)-Ethyl-4-Cyano-3-Hydroxybutyrate, rosuvastatin (CRESTOR™),atorvastatin (LIPITOR™), fluvastatin (LESCOL™) and their intermediates.Various starting materials can be chosen; cost may be a factor. Themethod can be in a whole cell process or a biocatalytic process or acombination thereof. At least one step in this exemplary method involvesuse of an enzyme. In alternative aspects, one, several or all steps usean enzyme.

The enzymatic reactions of the invention can be done in vitro or invivo, e.g., by whole cell methods. The enzymatic reactions can be donein vitro, including, e.g. capillary arrays, as discussed below, or, inwhole cell systems, also discussed further below. In one aspect, enzymereactions can be done in one or more reaction vessels. The enzymes orreagents of the invention can be immobilized onto solid surfaces, e.g.,arrays or capillary surfaces.

Whole Cell Engineering and Measuring Metabolic Parameters

The methods of the invention can be practiced in whole or in part in awhole cell environment. The invention also provides for whole cellevolution, or whole cell engineering, of a cell to develop a new cellstrain having a new phenotype to be used in the methods of theinvention, e.g., a new cell line comprising one, several or all enzymesof the invention, or an enzyme used in a method of the invention. Thiscan be done by modifying the genetic composition of the cell, where thegenetic composition is modified by addition to the cell of a nucleicacid, e.g., a coding sequence for an enzyme used in the methods of theinvention. See, e.g., WO0229032; WO0196551.

The host cell for the “whole-cell process” may be any cell known to oneskilled in the art, including prokaryotic cells, eukaryotic cells, suchas bacterial cells, fungal cells, yeast cells, mammalian cells, insectcells, or plant cells.

To detect the production of an intermediate or product of the methods ofthe invention (e.g., β,δ-dihydroxyheptanoic acid side chains and(R)-Ethyl-4-Cyano-3-Hydroxybutyrate), or a new phenotype, at least onemetabolic parameter of a cell (or a genetically modified cell) can bemonitored in the cell in a “real time” or “on-line” time frame byMetabolic Flux Analysis (MFA). In one aspect, a plurality of cells, suchas a cell culture, is monitored in “real time” or “on-line.” In oneaspect, a plurality of metabolic parameters is monitored in “real time”or “on-line.”

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

-   -   identity of all pathway substrates, products and intermediary        metabolites    -   identity of all the chemical reactions interconverting the        pathway metabolites, the stoichiometry of the pathway reactions,    -   identity of all the enzymes catalyzing the reactions, the enzyme        reaction kinetics,    -   the regulatory interactions between pathway components, e.g.        allosteric interactions, enzyme-enzyme interactions etc,    -   intracellular compartmentalization of enzymes or any other        supramolecular organization of the enzymes, and,    -   the presence of any concentration gradients of metabolites,        enzymes or effector molecules or diffusion barriers to their        movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.

Control of physiological state of cell cultures will become possibleafter the pathway analysis. The methods of the invention can helpdetermine how to manipulate the fermentation by determining how tochange the substrate supply, temperature, use of inducers, etc. tocontrol the physiological state of cells to move along desirabledirection. In practicing the methods of the invention, the MFA resultscan also be compared with transcriptome and proteome data to designexperiments and protocols for metabolic engineering or gene shuffling,etc. Any aspect of metabolism or growth can be monitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript orgenerating new transcripts in a cell. This increased or decreasedexpression can be traced by use of a fluorescent polypeptide, e.g., achimeric protein comprising an enzyme used in the methods of theinvention. mRNA transcripts, or messages, also can be detected andquantified by any method known in the art, including, e.g., Northernblots, quantitative amplification reactions, hybridization to arrays,and the like. Quantitative amplification reactions include, e.g.,quantitative PCR, including, e.g., quantitative reverse transcriptionpolymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or“real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001) Br. J. Haematol.114:313-318; Xia (2001) Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide or generatingnew polypeptides in a cell, e.g., enzymes of the invention (e.g., DERAenzymes) or other enzymes used in the methods of the invention. Thisincreased or decreased expression can be traced by use of a fluorescentpolypeptide, e.g., a chimeric protein comprising an enzyme used in themethods of the invention. Polypeptides, reagents and end products (e.g.,β,δ-dihydroxyheptanoic acid side chains or(R)-Ethyl-4-Cyano-3-Hydroxybutyrate) also can be detected and quantifiedby any method known in the art, including, e.g., nuclear magneticresonance (NMR), spectrophotometry, radiography (protein radiolabeling),electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, various immunological methods, e.g. immunoprecipitation,immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs),enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays,gel electrophoresis (e.g., SDS-PAGE), staining with antibodies,fluorescent activated cell sorter (FACS), pyrolysis mass spectrometry,Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, andLC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, andthe like. Novel bioactivities can also be screened using methods, orvariations thereof, described in U.S. Pat. No. 6,057,103. Polypeptidesof a cell can be measured using a protein array.

EXAMPLES Example 1 Measuring Aldolase Activity

In one aspect, an aldolase of the invention can convertfructose-1,6-diphosphate to dihydroxyacetone phosphate andglyceraldehyde-3-phosphate.

Measurement of serum aldolase activity can be of clinical significance.Elevated serum aldolase activity has been observed in certaincarcinomas, muscular dystrophy, hepatitis and myocardial infarction.Aldolases of the invention can be used for determination of metabolites,e.g., in coupled enzyme reactions.

Unit activity definition: the amount of enzyme which will convert onemicromole of fructose-1,6-diphosphate to dihydroxyacetone phosphate andglyceraldehyde-3-phosphate per minute at pH 7.6 and 25° C.

Assay reagents:

0.1 M Triethanolamine HCl buffer, pH 7.6.

0.008 M NADH, (5 mg/ml), NADH disodium salt in buffer.

0.033 M Fructose-1,6-diphosphate, (11.22 mg/ml) in buffer.

Glycerol-3-phosphate dehydrogenase (G3PDH) (76 U/ml) in buffer. Preparefresh.

Triose phosphate isomerase (TPI) (480 U/ml) in buffer. Prepare fresh.

Aldolase solution—dissolve in buffer to a final concentration of 0.1U/ml. Prepare fresh immediately prior to assay.

Protocol:

Set the spectrophotometer (equipped with a strip chart recorder andtemperature control) at 340 nm and 25° C.

Into a cuvette pipette the following:

Triethanolamine buffer 2.7 ml NADH solution 0.1 ml Fructose1,6-Diphosphate (substrate) 0.1 ml

Mix and incubate in the spectrophotometer at 25° C. for 5 min. toachieve temperature equilibration. Record blank at 340 nm, if any.

Add the enzyme solutions to the cuvette as follows:

Glycerol-3-phosphate dehydrogenase 0.01 ml Triose phosphate isomerase0.01 ml Aldolase  0.1 ml

Record the change in absorbance at 340 nm for 5-10 min.

Calculate Δ 304 nm/min

Kinetic Measurements Reactions were carried out in 0.1 M phosphatebuffer (pH 7.5) containing: varied concentrations of pyruvate, 2.0,3.33, 5, and 10 mM; varied concentrations of D-arabinose, 0.2, 0.25,0.33, and 0.50 M in 0.5 mL of solution. Each solution was incubated at37° C. The rates for aldolase-catalyzed reactions were obtained bymeasuring the amount of remaining pyruvate, according the method of Kim(1988) J. Am. Chem. Soc. 110:6481. Periodically, a small aliquot (25-100μL) was withdrawn and mixed with an assay solution (1.4 mL) containing0.1 M phosphate (pH 7.5) buffer, 0.3 mM NADH, and 20-30 U of L-lactatedehydrogenase. The decrease in absorbance at 340 nm was measured andconverted into the amount of the unreacted pyruvate using 6220 M⁻¹cm⁻¹for the molecular absorbance of NADH. The kinetic parameters wereobtained from the Lineweaver-Burk plots. For the relative ratemeasurements, the concentration of pyruvate (fluoropyruvate) and sugarwere fixed at 10 mM and 0.5 M, respectively. Other conditions were thesame as above.

Example 2 Conversion of3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone to(3R,5R)-6-cyano-3,5,-dihydroxyhexanoic acid

The invention provides processes for converting the lactone3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone (compound 1 of FIG.14, see also FIG. 17 and FIG. 18) in a single step to either(3R,5S)-3,5,6-trihydroxyhexanoic acid or(3R,5R)-6-cyano-3,5,-dihydroxyhexanoic acid (FIG. 16). The formercompound can be converted to rosuvastatin (CRESTOR™), fluvastatin(LESCOL™) and other statins, whereas the cyano compound can be convertedto atorvastatin (LIPITOR™). Both methods can go through a commonintermediate, the epoxide (-(3R,5S-3-hydroxy-4-oxiranylbutyric acidsodium salt) shown in brackets in FIG. 16.

In one aspect, an exemplary method for conversion of3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone to(3R,5R)-6-cyano-3,5,-dihydroxyhexanoic acid is: Sodium cyanide (8.93grams) was dissolved in 12 mL water. The aqueous solution was added to230 mL DMF, and 1 (10 grams) was added. Concentrations were 1 250 mM,NaCN 750 mM, water 5% by volume. The mixture was stirred at 40° C. for16 hours, concentrated under reduced pressure, then acidified to pH 4with sulfuric acid. NMR spectroscopy at a 6-hour timepoint showed thepresence of 35% (3R,5R)-6-cyano-3,5,-dihydroxyhexanoic acid product, 45%of the precursor (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid, and 20% of3R,5S-3-hydroxy-4-oxiranylbutyric acid. After 16 hours, the only productobserved was (3R,5R)-6-cyano-3,5,-dihydroxyhexanoic acid.

In one aspect, an exemplary method for conversion of 1 to(3R,5S)-3,5,6-trihydroxyhexanoic acid is: NaOH (176 mg, 2.2 equivalents)was dissolved in 5 mL water. 1 was added (330 mg, 400 mM concentration.The mixture was stirred at 40° C. for 16 hours, then concentrated underreduced pressure. NMR indicated complete conversion to(3R,5S)-3,5,6-trihydroxyhexanoic acid.

Example 3 Exemplary Process for the Synthesis of Statin Intermediates

One exemplary process for the synthesis of statin intermediates (for,e.g., synthesis of atorvastatin (LIPITOR™), rosuvastatin (CRESTOR™),fluvastatin (LESCOL™) and related compounds) is illustrated in FIG. 21,which is an exemplary aspect of the process illustrated in FIG. 14. Thefirst step comprises a DERA-catalyzed aldol condensation using, e.g., aDERA of the invention. 6-chloro-2,4,6-trideoxyerythro-hexonolactone isgenerated under conditions comprising aqueous NaOCl and HOAc. In oneaspect, the yield is greater than 99.9% ee, 45% over two steps. Themethod further comprises processing the3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone to make(3R,5R)-6-cyano-3,5,-dihydroxyhexanoic acid (see also, compound I ofFIG. 14). The last step of the process comprises dimethoxypropane,H₂SO₄, DMF and TMS diazomethane. The yield can be 48% over three steps.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An isolated, synthetic or recombinant nucleic acid comprising (a) anucleic acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more, or 100% sequence identity to SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 7, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ IDNO:29, over a region of at least about 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150 or more residues or the full length of a gene or atranscript, wherein the nucleic acid encodes at least one polypeptidehaving an aldolase activity, and the sequence identities are determinedby analysis with a sequence comparison algorithm or by a visualinspection; (b) a nucleic acid sequence encoding the amino acid sequenceof SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24,SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, or an enzymatically activefragment thereof; (c) a nucleic acid sequence that hybridizes understringent conditions to the nucleic acid of SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQID NO:29, wherein the nucleic acid encodes a polypeptide having analdolase activity, and the stringent conditions comprise a wash stepcomprising a wash in 0.2×SSC at a temperature of about 65° C. for about15 minutes (d) the nucleic acid sequence of (a), (b) or (c) encoding apolypeptide lacking a signal sequence; (e) the nucleic acid sequence of(a), (b), (c) or (d), further comprising a heterologous nucleic acidsequence or a heterologous nucleic acid sequence encoding a heterologouspeptide or polypeptide; (f) the nucleic acid sequence of (e), whereinthe heterologous nucleic acid sequence comprises a nucleic acid sequenceencoding a heterologous signal sequence (signal peptide), a heterologousprepro domain, a heterologous aldolase signal sequence (signal peptide)or a heterologous non-aldolase signal sequence (signal peptide); (g) thenucleic acid sequence of (e) or (f), wherein the heterologouspolypeptide or peptide is amino terminal to, carboxy terminal to or onboth ends of a signal peptide (SP), prepro domain or catalytic domain(CD); or (h) a nucleic acid sequence completely complementary to thenucleic acid sequence of (a), (b), (c), (d), (e), (f) or (g).
 2. Theisolated, synthetic or recombinant nucleic acid of claim 1, wherein thenucleic acid sequence encodes a polypeptide having a sequence as setforth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, or enzymaticallyactive fragments thereof.
 3. The isolated, synthetic or recombinantnucleic acid of claim 1, wherein the aldolase activity comprisescatalysis of the formation of a carbon-carbon bond; an aldolcondensation; a 2-deoxyribose-5-phosphate aldolase (DERA) activity;catalysis of the condensation of acetaldehyde as donor and a2(R)-hydroxy-3-(hydroxy or mercapto)-propionaldehyde derivative to forma 2-deoxysugar; catalysis of the condensation of acetaldehyde as donorand a 2-substituted acetaldehyde acceptor to form a 2,4,6-trideoxyhexosevia a 4-substituted-3-hydroxybutanal intermediate; catalysis of thegeneration of chiral aldehydes using two acetaldehydes as substrates;enantioselective assembling of chiral β,δ-dihydroxyheptanoic acid sidechains; enantioselective assembling of the core of[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, or LIPITOR™); rosuvastatin (CRESTOR™) or fluvastatin(LESCOL™); with an oxidation step synthesis of a3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone; a thermotolerantaldolase activity; or, a thermostable aldolase activity.
 4. A nucleicacid probe for identifying a nucleic acid encoding a polypeptide with analdolase activity, wherein the probe comprises at least 10, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive bases of asequence comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQID NO:29, wherein the probe identifies the nucleic acid by binding orhybridization, wherein the stringent conditions comprise a wash stepcomprising a wash in 0.2×SSC at a temperature of about 65° C. for about15 minutes. 5-10. (canceled)
 11. A vector, a cloning vehicle or anexpression cassette comprising (a) a nucleic acid comprising the nucleicacid sequence of claim 1; or (b) the vector, cloning vehicle orexpression cassette of (a) comprising or consisting of a viral vector, aplasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or anartificial chromosome.
 12. A transformed cell comprising a nucleic acidcomprising the sequence of claim 1, or the vector, cloning vehicle orexpression cassette of claim
 11. 13. A transgenic non-human animalcomprising a nucleic acid comprising the sequence of claim 1, or thevector, cloning vehicle or expression cassette of claim
 11. 14. Atransgenic plant or seed comprising a nucleic acid comprising thesequence of claim 1, or the vector, cloning vehicle or expressioncassette of claim
 11. 15-18. (canceled)
 19. An isolated, synthetic orrecombinant polypeptide comprising: (i) an amino acid sequence having atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or is 100%sequence identity to SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, orenzymatically active fragments thereof, over a region of at least about100, 150, 200, 250, 300, 350, 400, 450, 500 or more residues, whereinthe sequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection, and the polypeptide hasan aldolase activity; (ii) an amino acid sequence encoded by the nucleicacid of claim 1, wherein the polypeptide has aldolase activity; (iii)the amino acid sequence of (i) or (ii) lacking a signal sequence; (iv)the amino sequence of (i), (ii) or (iii), further comprising aheterologous peptide or polypeptide; (v) the amino acid sequence of(iv), wherein the heterologous peptide or polypeptide comprises aheterologous signal sequence (signal peptide), a heterologous preprodomain, a heterologous aldolase signal sequence (signal peptide) or aheterologous non-aldolase signal sequence (signal peptide); (vi) theamino sequence of (iv) or (v), wherein the heterologous polypeptide orpeptide is amino terminal to, carboxy terminal to or on both ends of asignal peptide (SP), prepro domain or catalytic domain (CD); or (vii)the amino acid sequence of (i), (ii), (iii), (iv), (v) or (vi), whereinthe aldolase activity comprises catalysis of the formation of acarbon-carbon bond; an aldol condensation; a 2-deoxyribose-5-phosphatealdolase (DERA) activity; catalysis of the condensation of acetaldehydeas donor and a 2(R)-hydroxy-3-(hydroxy or mercapto)-propionaldehydederivative to form a 2-deoxysugar; catalysis of the condensation ofacetaldehyde as donor and a 2-substituted acetaldehyde acceptor to forma 2,4,6-trideoxyhexose via a 4-substituted-3-hydroxybutanalintermediate; catalysis of the generation of chiral aldehydes using twoacetaldehydes as substrates; enantioselective assembling of chiralβ,δ-dihydroxyheptanoic acid side chains; enantioselective assembling ofthe core of[R—(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-(phenylamino)-carbonyl]-1H-pyrrole-1-heptanoicacid (atorvastatin, or LIPITOR™); rosuvastatin (CRESTOR™) or fluvastatin(LESCOL™); with an oxidation step synthesis of a3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone; a thermotolerantaldolase activity; or, a thermostable aldolase activity.
 20. (canceled)21. A composition comprising the polypeptide of claim
 19. 22. Aheterodimer or a homodimer comprising the polypeptide of claim 19 and asecond domain. 23-25. (canceled)
 26. An isolated, synthetic orrecombinant antibody that specifically binds to the polypeptide of claim19. 27-30. (canceled)
 31. A method of producing a recombinantpolypeptide comprising the steps of: (a) providing a nucleic acidoperably linked to a promoter, wherein the nucleic acid comprises thesequence of claim 1; and (b) expressing the nucleic acid of step (a)under conditions that allow expression of the polypeptide, therebyproducing a recombinant polypeptide. 32-41. (canceled)
 42. A method ofgenerating a variant of a nucleic acid encoding a polypeptide with analdolase activity comprising the steps of: (I)(a) providing a templatenucleic acid comprising the sequence of claim 1; and (b) modifying,deleting or adding one or more nucleotides in the template sequence, ora combination thereof, to generate a variant of the template nucleicacid or (II) (a) providing a nucleic acid encoding a polypeptide with analdolase activity comprising the sequence of claim 1; and, (b)identifying a non-preferred or a less preferred codon in the nucleicacid of step (a) and replacing it with a preferred or neutrally usedcodon encoding the same amino acid as the replaced codon. 43-48.(canceled)
 49. An isolated, synthetic or recombinant signal sequence(signal peptide, or SP) consisting of a sequence as set forth inresidues 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22,1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30or 1 to 31, 1 to 32 or 1 to 33 of SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30, or, residues 1 to 22 of SEQ ID NO:18.
 50. A chimeric polypeptidecomprising at least a first domain comprising a signal sequence (signalpeptide, or SP) having the sequence of claim 49, and at least a seconddomain comprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal sequence (signal peptide, or SP).
 51. (canceled)
 52. A method ofincreasing thermotolerance or thermostability of an aldolasepolypeptide, the method comprising glycosylating an aldolase, whereinthe polypeptide comprises at least thirty contiguous amino acids of thepolypeptide of claim 19, or a polypeptide encoded by the nucleic acid ofclaim 1, thereby increasing the thermotolerance or thermostability ofthe aldolase.
 53. A method for overexpressing a recombinant aldolase ina cell comprising expressing a vector comprising the nucleic acid ofclaim 1, wherein overexpression is effected by use of a high activitypromoter, a dicistronic vector or by gene amplification of the vector.54. A method of making a transgenic plant comprising the followingsteps: (a) introducing a heterologous nucleic acid sequence into a plantcell, wherein the heterologous nucleic sequence comprises the sequenceof claim 1, thereby producing a transformed plant cell; (b) producing atransgenic plant from the transformed cell.
 55. (canceled)
 56. A methodfor preparation of a compound having a formula as set forth asintermediate II in FIG. 7, comprising the following steps: (a) providingan aldol donor substrate; (b) providing an aldol acceptor substrate; (c)providing an aldolase; (d) admixing the aldol donor substrate of step(a), the aldol acceptor substrate of step (b), and the aldolase of step(c) under conditions wherein the aldolase can catalyze the aldolcondensation reaction between the substrates of steps (a) and (b)thereby producing a compound comprising a structure as set forth asintermediate II in FIG.
 7. 57. A process for making atorvastatin(LIPITOR™) comprising a process as set forth in FIG.
 14. 58. A processfor making rosuvastatin (CRESTOR™) or fluvastatin (LESCOL™) comprising aprocess as set forth in FIG. 14 or FIG.
 17. 59. A method for preparationof a compound having a formula as set forth as intermediate II in FIG.7, using a fed-batch process, comprising the following steps: (a)providing an aldol donor substrate; (b) providing an aldol acceptorsubstrate, (c) providing an aldolase; (d) admixing the aldol donorsubstrate of step (a), the aldol acceptor substrate of step (b), and thealdolase of step (c) under conditions wherein the aldolase can catalyzethe aldol condensation reaction between the substrates of steps (a) and(b), wherein the substrates are fed into the reaction over about atleast about 30 minutes to 12 hours at a rate such that they are consumedas fast as they are added.
 60. A method for making3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone (compound 1 of FIG.14) comprising oxidation of a chlorolactol to a chlorolactone withsodium hypochlorite.
 61. A method for making3R,5S-6-chloro-2,4,6-trideoxy-erythro-hexonolactone (compound 1 of FIG.14) comprising a process as set forth in FIG.
 15. 62. A method formaking an epoxide (-(3R,5S-3-hydroxy-4-oxiranylbutyric acid) (structure2 in FIG. 16) comprising use of NaCN, DMF and 5% H₂O.
 63. A method formaking (3R,5S)-3,5,6-trihydroxyhexanoic acid comprising a process as setforth in FIG.
 16. 64. (canceled)